Name: indirect immunofluorescence on frozen sections of mouse mammary gland
Uploaded: 2015-12-01T14:00:00.000+00:00
Duration: 11 min 13 s
Description: Watch this Scientific Journal Video about Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland at JoVE.com

The authors are grateful to the INRA MIMA2 imaging core facility (INRA, UMR1198, Jouy-en-Josas) and to the staff of the IERP unit (UE 0907, INRA, Jouy-en-Josas) for animal care and facilities. We would also like to thank I.H. Mather, M.C. Neville and S. Tooze for providing us with very useful antibodie.

CD1 mice were bred at INRA (UE0907 IERP, Jouy-en-Josas, France). All ethical aspects of animal care complied with the relevant guidelines and licensing requirements laid down by the French Ministry of Agriculture. The procedures used were approved by the local ethics committee (agreement 12/097 from the Comethea Jouy-en-Josas/AgroParisTech).
1. Mammary Gland Sample Preparation
<ol>
	<li>Mouse mammary gland dissection
	<ol>
		<li>Euthanize mice at day 10 of lactation by cervical dislocation and pin the animal down with its abdomen facing up.&#160;</li>
		<li>Wet the ventral area with ethanol and dry it with a paper towel.&#160;</li>
		<li>Using forceps, pull up the abdominal skin between the two hind legs and make an incision (through the skin only) of about 1 cm with sharp scissors. Starting from this first incision, then use scissors to cut the skin up to the neck on the mouse. Pull the skin away from the peritoneum and pin down one side of the skin at a time, stretching it taught.&#160;</li>
		<li>Collect the abdominal and the inguinal mammary glands by pushing them away from the skin with a swab and finally pulling or cutting them away from the peritoneum.&#160; 
		Note: At this step Carmine staining can be performed in order to visualize the mammary epithelium within the entire gland43. This approach can be useful to analyze the global morphology of the mammary gland under various conditions (physiological developmental stages, diseases, in vivo treatments).</li>
		<li>Remove the lymph node located at the junction of the abdominal and the inguinal glands44.</li>
	</ol>
	</li>
	<li>Mammary tissue fixation
	<ol>
		<li>Cut the mammary tissue into 3 mm3 fragments with a scalpel and immediately rinse these fragments in a phosphate buffered saline (PBS) solution, pH 7.4, in order to remove as much milk as possible.&#160;</li>
		<li>Quickly dry the fragments on a paper towel and put them in a cold PBS solution containing 4% paraformaldehyde (PFA, HCHO, 32% formaldehyde solution, CAUTION) for 10 to 15 min on ice. 
		Note: This is enough time to allow subsequent analysis on mammary tissue slices by IIF36 and/or in situ hybridization45. However, as aldehyde fixatives penetrate rather slowly in tissue pieces (~1-3 mm per hour), this time may be extended to ensure an optimal fixation of the tissue sample. Alternatively, fix tissues in vivo by perfusing an anesthetized animal with a fixative solution (not detailed in the present study).</li>
	</ol>
	</li>
	<li>Sucrose infusion
	<ol>
		<li>Quickly rinse the mammary fragments in cold PBS and immerse them in a cold PBS solution containing 40% sucrose (D-saccharose, C12H22O11, Mr 342.3 g/mol) for 16 to 48 hr at 4 &#176;C under gentle shaking.</li>
	</ol>
	</li>
	<li>Tissue embedding 
	Note: At this step, mammary fragments can be re-cut in order to make smaller fragments (2-3 mm3) or to adjust their shape.&#160;
	<ol>
		<li>Properly label the plastic molds and fill a third of the volume of the mold with OCT compound, maintained at RT. Place one fragment (2-3 mm3) of mammary tissue per mold and cover it with OCT compound.&#160;</li>
		<li>Place the molds at the surface of the liquid nitrogen (on a sheet of aluminum or using a metallic sieve) and allow the product to freeze. 
		Note: It must become solid and white before immersing the mold in liquid nitrogen.</li>
	</ol>
	</li>
	<li>Store the frozen samples at -80 &#176;C until tissue sections are performed.</li>
</ol>
2. Frozen tissue Sectioning
Note: A cryostat, which is essentially a microtome inside a freezer, is required to make frozen tissue sections. A lower temperature is often required for fat or lipid-rich tissues such as virgin mammary gland.
<ol>
	<li>Adjust the temperature of the cryostat to -26 &#176;C and wait until it has stabilized. Maintain the frozen tissue block at -26 &#176;C throughout the entire sectioning procedure. Absolutely avoid thawing the tissue at any time during the procedure.</li>
	<li>Cool the razor blade, the cutting support, the anti-roll device and the brush to -26 &#176;C by placing them in the cryostat for at least 10 min. Also place a slide box inside the cryostat in order to be able to store glass slides as the sections are made.&#160;</li>
	<li>Properly label the glass slides that will be used to collect the tissue sections and maintain them at RT; otherwise tissue sections will not adhere to them. Remove the sample from the mold inside the cryostat.&#160; 
	Note: Using positively charged glass slides will greatly favor the adhesion of fresh frozen tissue sections due to higher electrostatic attraction.</li>
	<li>Cover the surface of a metal tissue disc with OCT compound (maintained at RT) and push the frozen sample onto it. Place the wet mount inside the cryostat and let it cool for at least 15 min.</li>
	<li>Place the wet mount in the disc holder of the cryostat. Adjust the cut thickness to 5-6 &#181;m and, if possible, use a new sharp blade or at least change the area on the blade used to cut each sample since some tissues will quickly dull it.&#160;</li>
	<li>Adjust the position of the anti-roll device over the razor blade by making cuts of the mounting medium until the slices are formed evenly and correctly. Ideally, the anti-roll device will step over the razor blade by about 1 mm.&#160;</li>
	<li>Once the settings are correct, perform tissue sections by turning the wheel in a continuous uniform motion. Unless the temperature is ideal, a tissue section will, by nature, try to curl up.&#160;
	<ol>
		<li>Use a brush to grab and maneuver the section across the stage in order to place it as desired on the glass slide. Use the brush to clean up the remains possibly present on the frozen tissue block and/or the razor blade.&#160;</li>
	</ol>
	</li>
	<li>Pull the tissue section toward the user and avoid pressing it onto the cryostat stage. Avoid pressing the tissue section onto the cryostat stage as it may lead to the adhesion of the tissue slice on the stage and thus the inability to recover it with the glass slide.</li>
	<li>Retrieve tissue sections one by one by picking them up at the surface of a glass slide, by holding it above the section and angling it down to touch the tissue section. 
	Note: Tissue sections quickly adhere to the warm glass due to static attraction. If several tissue sections are placed on the same slide, be careful not to overlap them and to space them enough to be able to individually enclose them in a hydrophobic circle (see section 3.1.1.).</li>
</ol>
3. Indirect Immunofluorescence
<ol>
	<li>Locating sections
	<ol>
		<li>Use a hydrophobic barrier pen to draw a hydrophobic circle around slide-mounted tissue. Let the circle dry for approximately 1 min at RT. Draw a line around the tissue sections with a fine black permanent marker as well, but on the side of the glass slide opposite to the one where the tissue sections are.&#160; 
		Note: This circle is water-repellent and acetone- and alcohol-insoluble. It therefore provides a barrier to aqueous solutions used during the IHC procedure and reduces the volume of required reagents.&#160;</li>
		<li>Rehydrate tissue sections by covering them with a drop of ~250 &#181;l of PBS for a few minutes at RT. Fix tissue sections by covering them with ~250 &#181;l of a freshly prepared 3% PFA solution in PBS for 10 to 15 min.&#160; 
		Note: Optionally in this case, use an aldehyde quenching solution (50 mM ammonium chloride (NH4Cl, Mr 53.5 g/mol) in PBS or 0.1M glycine (C2H5NO2, Mr 75.07 g/mol) in PBS) to stop the fixation reaction. Simple and abundant PBS washing is generally sufficient to remove unreacted aldehyde.</li>
	</ol>
	</li>
	<li>Antigen retrieval (optional) 
	Note: While most antigens can be detected without antigen retrieval (AR), others will be observed only if this step is performed. In some cases, AR also allows the enhancement of the observed signal.
	<ol>
		<li>Place the AR solution (100 mM Tris (C4H11NO3, Mr 121.14) 5% urea (NH2CONH2, Mr 60.06) pH 9.6) in a beaker. The volume of AR solution must be sufficient to completely cover the glass slides placed in a glass holder.&#160;</li>
		<li>Preheat the AR solution to 95 &#176;C by monitoring the temperature with a thermometer and then place the glass slides on a suitable rack, immerge the rack in the hot buffer, cover to limit evaporation and incubate for 10 min at 95 &#176;C.&#160;</li>
		<li>Remove the beaker from the water bath and leave the glass slides for another 10 min in the buffer.</li>
	</ol>
	</li>
	<li>Immunodetection
	<ol>
		<li>Rinse tissue sections with PBS (~250 &#181;l/section) and saturate them with a solution of 3% bovine serum albumin (BSA, ~250 &#181;l/section) in PBS for at least 30 min at RT.&#160;</li>
		<li>Put 30-50 &#181;l of the primary antibody diluted in PBS containing 2% BSA on each tissue section. 
		Note: This volume is enough to form a drop that completely covers the tissue section.</li>
		<li>Place the same volume of the diluent (2% BSA in PBS) alone on a tissue section to perform a negative control without primary antibody.
		<ol>
			<li>Systematically include this negative control in each IHC experiment and perform for each secondary antibody used to estimate the background of the experiment (non-specific labeling due to the secondary antibody and/or the tissue auto-fluorescence). Other types of positive or negative controls can also be performed to ensure the specificity of the labeling (see discussion).</li>
		</ol>
		</li>
		<li>Place the glass slides in a humidified box O/N at 4 &#176;C. 
		Note: Primary antibodies used were mouse monoclonal anti-cytokeratin 8 (CK8, 1:50 dilution), mouse monoclonal anti-cytokeratin 14 (CK14, 1:50 dilution), rabbit polyclonal anti-mouse casein (#7781, 1:50 dilution, generously provided by M.C. Neville, University of Colorado Health Sciences Center, CO, USA), rabbit polyclonal anti-BTN1 (1:300 dilution, generously provided by I.H. Mather, Department of Animal and Avian Sciences, University of Maryland, College Park, MD, USA), rabbit polyclonal anti-Stx6 (1:50 dilution, generously provided by S. Tooze, Cancer Research UK, London Research Institute, London, UK) and rabbit polyclonal anti-VAMP4 (1:50 dilution).&#160;</li>
		<li>Thoroughly wash tissue sections with PBS at least four times for 10 min at RT.</li>
		<li>Dilute the appropriate secondary antibody (rhodamine-conjugated goat anti-rabbit IgG (H + L), 1:300 dilution) in PBS containing 2% BSA, place 30-50 &#181;l of this solution on all tissue sections, and incubate for 1.5 hr at RT.
		<ol>
			<li>Since fluorochromes are light-sensitive molecules, do not expose tissue sections to light until their analysis. For IIF on tissue sections, favor secondary antibodies coupled to a red fluorophore since cell membranes tend to generate a green auto-fluorescence that can interfere with low labeling. Moreover, choosing a red fluorophore-coupled secondary antibody allows the concomitant labeling of neutral lipids (see below).&#160;</li>
		</ol>
		</li>
		<li>Thoroughly wash the tissue sections with PBS at least four times for 10 min at RT.&#160;</li>
	</ol>
	</li>
	<li>Post-fixation (optional)
	<ol>
		<li>For some experiments, perform post-fixation by incubating the samples with 2% PFA diluted in PBS for 10 min at RT in order to stabilize the antigen/antibody scaffolds. However, this step can be dispensed with in most cases.</li>
	</ol>
	</li>
	<li>Neutral lipids and DNA counterstaining
	<ol>
		<li>To visualize CLDs and MFGs, color neutral lipids by incubating tissue sections in 30-50 &#181;l of a PBS solution containing 3 &#181;g/ml of bodipy 493/503 for 10 min at RT. Rapidly rinse tissue sections twice with PBS.</li>
		<li>Counterstain nuclear DNA with 30-50 &#181;l of a PBS solution containing 3 &#181;M of DAPI (4-6-diamidino-2-phenylindole, 5 mg/ml stock solution) for 10 min at RT. Wash the tissue sections twice with PBS before mounting the slides for observation.</li>
	</ol>
	</li>
	<li>Section mounting
	<ol>
		<li>Remove PBS and place a drop of mounting medium on each tissue section.&#160;</li>
		<li>Place one side of the cover slip at an angle against the slide, making contact with the outer edge of the liquid drop and then lower the cover slowly, avoid air bubbles. Allow the liquid to spread between the glass slide and the cover slip for a few minutes and then remove the excess of mounting medium with a paper towel.</li>
		<li>Seal the cover slip to the glass slide with nail polish and store tissue sections at 4 &#176;C to prevent their exposure to light until observation.</li>
	</ol>
	</li>
</ol>
4. Fluorescence Observation and Image Acquisition
Note: A fluorescence microscope equipped with a camera controlled by image acquisition software is required to observe the IHC results.
<ol>
	<li>Before acquiring images, check the intensity of the labeling and evaluate the background of the experiment by looking at the negative controls. Acquire pictures of each fluorescent label (color channel) individually.</li>
	<li>Acquire all pictures, including those of the corresponding controls, in the same conditions (exposure and general settings) for each color channel.</li>
	<li>Conventional microscopy
	<ol>
		<li>Perform epifluorescence microscopy with a microscope equipped with standard filters for fluorescein isothiocyanate (FITC, green), rhodamine (red) and DAPI (blue) emissions, &#215;20 to &#215;63 (oil-immersion, NA 1.3) objectives and a DP50 imaging camera.</li>
	</ol>
	</li>
	<li>Confocal microscopy</li>
	<li>Perform confocal microscopy with a microscope equipped with the ZEN software, using &#215;20 to &#215;63 (oil-immersion, NA 1.4) objectives and the 488- and 568-nm excitation wavelengths of the laser.</li>
</ol>
5. Image Treatment
Note: All image post-treatments are performed using the ImageJ free software (http://imagej.nih.gov/ij/).
<ol>
	<li>Superimpose image (merge)
	<ol>
		<li>Open the images acquired in each channel that will be combined (File/Open). If working with 8-bit grayscale images, attribute artificial color to each channel using the lookup table (Image/Lookup Tables).&#160;</li>
		<li>Generate the composite picture from grayscale or colored images by using the command &#8220;Merge channels&#8221; (Image/Color/Merge Channels) and then attributing a color to each channel.&#160;</li>
		<li>Perform image stacks superimposition in the same way by opening stacks acquired in each channel that will be combined (File/Open) and using the command &#8220;Merge channels&#8221; (Image/Color/Merge Channels) to attribute a color to each channel. Save the composite stack as an image sequence or as a movie (see section 5.4).</li>
	</ol>
	</li>
	<li>Image stack Z projection
	<ol>
		<li>Use the Z projection function (Image/Stack/Zproject, Max Intensity) to provide a two-dimensional view of all the pictures of an image stack by projecting them along the axis perpendicular to the image plane (z-axis). The &#8220;Maximum Intensity&#8221; option creates an image in which each pixel contains the maximum value over all images in the stack. This generates a single image allowing the visualization of all the staining observed through the whole image stack for a particular channel or after the superimposition of several channels.</li>
	</ol>
	</li>
	<li>Image stack 3D projection
	<ol>
		<li>Use the 3D projection command (Image/Stack/3D project, Brightest Point, y-axis) to generate a sequence of projections of a rotating volume onto a plane. The visual rendering of surfaces and internal structures depends on both the projection method (nearest-point, brightest point (used here), or mean-value) and the visualization parameters selected. Each frame of the animated sequence is the result of projecting from a different viewing angle.&#160;</li>
		<li>Rotate the created 3D image around each of the three orthogonal axes (the y-axis was selected here). Save the sequence produced as a single image or a movie.</li>
	</ol>
	</li>
	<li>Image stack to movie conversion
	<ol>
		<li>Open an image stack (File/Open) and save it as a movie in .AVI format by using the command &#8220;AVI&#8221; (File/Save As/AVI).</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests.

IHC is a relatively simple and straightforward experimental method to localize antigen in tissue sections, which depends primarily on specific epitope-antibody interactions. Although a large number of protocols are used to localize a protein by IIF, the core of these procedures is almost always the same. However, there are some critical aspects that can strongly influence the result and must therefore be optimized for each individual IHC study. The most challenging aspect of this approach is to determine the best experimental conditions, i.e., those generating a strong and specific signal for the antigen of interest. The variables that must be considered for experimental design and optimization are: (1) the type of antigen (species, expression levels, subcellular location); (2) the epitope type (sequence, conformation, putative post-translational modifications); (3) sample preparation (embedding in paraffin or for frozen sections); (4) the fixation method (perfusion or immersion); (5) the fixative used (formaldehyde, alcohol or acetone); (6) the blocking reagent used (normal serum, BSA or non-fat milk); (7) the AR step; (8) the detection method (direct or indirect); (8) the type of primary antibody (monoclonal or polyclonal); (9) the secondary antibody (species and label); (10) counterstains (nuclear and/or other cellular compartment labeling); and (11) the mounting medium (see Table 1 for details). The fixation and the blocking steps, at least, require the optimization of additional factors such as concentration, pH, temperature, incubation time and diluent.

The first crucial aspect concerns the preparation of tissue samples, which is closely linked to the fixation method, which in turn influences the quality of the results. For example, tissue pieces can be fixed or not prior to embedding. This step may also depend on the embedding method chosen, i.e., OCT compound vs. paraffin embedding, which itself sometimes depends on the primary antibody used. Tissue fixation can be performed in vivo by perfusing an anesthetized animal with a fixative solution. This method is useful to preserve antigens when studying intact tissues but may not be sufficient to fix the tissue of interest. In this case, small tissue pieces (no thicker than 10 mm) can be immersed in the fixative solution. Frozen tissue can be prepared by immersing the tissue in liquid nitrogen or isopentane, and snap-freezing is highly recommended for the subsequent detection of post-translational modifications such as phosphorylation. However, unlike paraffin-embedded tissue, freezing is not adequate for long-term preservation of the tissues due to the formation of ice crystals within the cells that may alter subcellular morphology. Once cut, frozen tissue sections can be stored at -80 &#176;C for up to 1 year. In any case, the preparation of the tissue samples is a compromise between preserving the tissue/cellular architecture and preserving the epitope integrity.

Since it alters the chemical composition of tissues, it is critical to optimize the fixation conditions to avoid both incomplete (under&#64257;xation) and excessive (overfixation) &#64257;xation.
Indeed, underfixation may reduce the specific signal by promoting the proteolytic degradation of certain antigens. On the other hand, overfixation can alter the specific labeling by masking the epitope or generating a strong non-specific background. Thus, in addition to the choice of the fixative solution, other parameters such as the incubation time, the temperature and the pH will affect tissue fixation. Although PFA is the most common fixative used for IHC, it cannot be considered as a &#8220;universal&#8221; fixative. PFA induces protein-protein and protein-nucleic acid cross-links and can thus artefactually modify the epitope (overfixation) and then prevent its recognition by the primary antibody. However, the epitope can be further unmasked by AR techniques (see below). PFA may also be unsuitable for the detection of certain antigens, as it has been shown to induce the translocation of some phosphorylated proteins from the membrane to the cytoplasm. In such cases, PFA must be replaced by appropriate alternative fixatives such as alcohol. Unlike PFA, alcohols such as methanol or ethanol do not mask epitopes since they allow tissue fixation by replacing water molecules in tissues. This can lead to the precipitation of the proteins and then prevent antibody/epitope interaction due to conformational changes. It is widely thought that alcohols do not penetrate and thus do not preserve tissue morphology as well as PFA. Acetone is another alternative fixative, which is commonly used when working with unfixed, snap-frozen tissue sections. However, acetone is a strong dehydrating agent and can lead to irreversible precipitation of tissue proteins.

For some antigens, an additional step of AR may be required to obtain a good signal, mainly if the fixative induces the conformational change or alters the electrostatic charge of the epitope (masking of the epitope). AR methods aim to reverse these processes to restore the immunoreactivity of the epitope and its subsequent interaction with the primary antibody. AR methods mainly rely on two approaches: (1) protease-induced epitope retrieval, i.e., with enzymes such as proteinase K, trypsin or pepsin, which cleave peptides that mask the epitope; and (2) heat-induced epitope retrieval, i.e., using a microwave oven, pressure cookers, vegetable steamers, autoclaves or water baths. This latter approach is especially time-, temperature-, buffer-, and pH-sensitive, and the optimal conditions must be empirically determined (an example is provided in the Protocol section). Alternatively, the affinity of an antibody for its antigen can be enhanced by changing the pH or the cation concentration of the antibody diluent.

A permeabilization step is sometimes required to obtain a good signal for an intracellular epitope in thick tissue sections, particularly for nuclear antigen staining. This can be achieved in various ways by using: (1) alcohols or acetone as fixatives; or (2) detergents such as Triton, NP-40 (0.1-0.2% in PBS, 10 min), digitonin, saponin or Tween 20 (0.2-0.5% for 10 to 30 min) after PFA fixation. However, the choice of the detergent depends on the cellular location of the epitope detected. Indeed, harsh detergents such as Triton-X100, which solubilize cellular membranes, are suitable for nuclear epitope detection but can lead to signal alteration upon the extraction of some membranous proteins. The use of milder detergents (saponin and Tween 20) are more suitable for the detection of cytoplasmic epitopes.

The second critical step is the blocking of non-specific staining. The binding of an antibody to its target epitope is governed by intermolecular forces (e.g., hydrophobic and ionic interactions, hydrogen bonding). Thus, interactions of primary and/or secondary antibodies with other proteins than their target antigens may result in non-specific staining. This generates high fluorescence background, which prevents the visualization of the protein of interest (low signal/noise ratio). Blocking reagents reduces non-specific interactions without impairing specific antibody/epitope interaction. A common procedure consists of incubating tissue sections with heat-inactivated normal serum or BSA. When using a normal serum, it must be from the same species as that of the host animal of the secondary antibody or from an unrelated species. In all cases, the selected blocking reagent must also be added to the diluents for the primary and secondary antibodies. Furthermore, the use of non-ionic detergents such as Triton X-100, Tween 20 or saponin helps to reduce non-specific interactions.

The third and probably most important parameter is the primary antibody selection and optimization. Obviously, the best choice is a high-quality antibody with minimal cross-reactivity. As monoclonal antibodies usually exhibit high affinity and specificity for a single epitope, they are the best tools to discriminate a particular member of a protein family with high sequence identity. However, the antibody/epitope interaction can be compromised if the target epitope has lost its native conformational state or when the access to the epitope is prevented by interactions with other proteins, post-translational modifications, temperature, pH, fixation and salt concentration. In such cases, polyclonal antibodies are more suitable as they recognize multiple epitopes of the same protein. Moreover, they are often more stable than monoclonal antibodies over a wide range of pH and salt concentration. Preliminary studies have to define the appropriate incubation conditions, i.e., working dilution (monoclonal antibody: 5-25 mg/ml, polyclonal antibody: 1.7-15 mg/ml), incubation time, diluent and temperature, which have to be empirically determined for each primary antibody. These parameters have to be optimized to determine the conditions that produce the optimal signal with low background noise. The specificity of the labeling is favored by longer incubation times at lower temperatures (i.e., 4 &#176;C vs. RT).

The choice to perform direct or indirect detection often depends on the level of antigen expression. For example, a highly expressed epitope can simply be detected with a fluorochrome-conjugated primary antibody, thus allowing a fast and simple multicolor staining while avoiding possible non-specific background due to the use of a secondary antibody. However, direct IF may generates a low signal at a higher cost, and can sometimes be difficult, when labeled antibody are not commercially available. Conversely, IIF is more sensitive to detect lower expressed epitopes as the generated signal is more intense due to the interaction of at least two labeled secondary antibodies (raised against the primary antibody host species) with the primary antibody (amplification). Furthermore, a wide range of secondary antibodies conjugated to various fluorophores are commercially available, relatively inexpensive, and quality controlled. However, this approach can induce cross-reactivity and thus requires to carefully choose primary antibodies that are not produced in the same species or of different isotypes when performing multiple-labeling experiments. IIF also sometimes requires extra blocking steps and must include systematic negative controls (see below). Amplification can be further achieved by using a biotin-conjugated secondary antibody and fluorescently labeled avidin or streptavidin (four biotins bound per molecule). Nevertheless, this amplification method requires additional steps to prevent non-specific binding and may not be adapted for the staining of some tissues (liver, kidney, heart, brain, lung and lactating mammary gland) due to the presence of high levels of endogenous biotin. However, endogenous biotin can be blocked by pre-incubating the sample with avidin and subsequently with biotin before incubation with the primary antibody. The choice of the conjugated fluorochromes, which are small chemical molecules with the property to emit light when excited by light of a shorter wavelength, primarily depends on the type of microscope equipment available.

When properly designed to limit both cross-reactivity between the antibodies and crossover between the spectral properties of the fluorochromes used, immunofluorescence-based IHC allows the simultaneous visualization of multiple cellular targets.

The last critical point regarding IHC experiments concerns the positive and negative controls that must be performed to support the validity of staining, to identify experimental artifacts and for the accurate interpretation of the results. Some tissues exhibit high fluorescent background (referred to as autofluorescence) that could lead to a misinterpretation of the results. Thus, tissue sections have to be observed under both fluorescence and bright-field illumination before starting the IHC experiment. A negative control that omits the primary antibody must systematically be included in each IHC experiment in order to ensure that a potential non-specific binding of the secondary antibody is negligible and does not obscure or resemble the specific staining pattern. An isotype control can be performed when working with a monoclonal primary antibody by replacing it with a non-immune antibody of the same isotype (e.g., IgG1, IgG2A, IgG2B, IgM) at the same concentration. This control helps to estimate the non-specific staining, which may be due to the interactions of antibodies with the sample. To demonstrate the specific binding of an antibody to its antigen, an absorption control can be achieved in two ways by pre-incubating the primary antibody: (1) with its soluble immunogen (10:1 molar ratio) O/N at 4 &#176;C; and (2) with cells or tissue sections that express the epitope of interest but that differ from the studied tissue (for example, see Figure 4B in59). In both cases, the consequent depletion of the primary antibody should lead to little or no staining. Another type of control can be done using an irrelevant primary antibody, i.e., directed against an epitope that exhibits a cellular localization that is different from the epitope of interest (nuclear vs. cytoplasmic). The irrelevant antibody must be of the same isotype and species as the primary antibody of interest. Additional controls for IHC experiments can include the use of samples from tissues known to express (transgenic animals) or not (knock-out animals) the epitope of interest. This can provide a useful reference and help to optimize the IHC procedure.

A main limitation of IF techniques is that they can only be applied to fixed (dead) and/or permeabilized cells, both procedure potentially inducing artifacts. Other limitations of this approach are due to the use of a microscope for the observation of the samples. First, as the optical resolution of epifluorescence and confocal microscopes is limited, location or co-location of the detected proteins should not be over-interpreted. Secondly, photobleaching, i.e. fading of the fluorescence intensity over time when exposed to light, is essentially due to generation of reactive oxygen species in the sample upon fluorescence excitation which, in turn, leads to the photochemical destruction of the fluorophore. Photobleaching can be reduced by: a) keeping the samples protected from light during the IF experiment and storage until their observation; b) using an antifade agent (reactive oxygen species scavengers) in the mounting medium; c) reducing the intensity and/or duration of the excitation light; d) increasing the concentration of fluorophores or using a low concentration of a fluorochrome with high-quantum efficiency; and e) using robust fluorophores that are less prone to photobleaching (i.e. Alexa Fluors, Seta Fluors, or DyLightFluors). Thirdly, autofluorescence is often due to the presence of flavin coenzymes (FAD and FMN: absorption, 450 nm; emission, 515 nm) and reduced pyridine nucleotides (NADH: absorption, 340 nm; emission, 460 nm) in mammalian cells. Furthermore, the use of aldehydes, particularly glutaraldehyde, to fix the samples, can result in high levels of autofluorescence. This can be minimized by washing the samples with 0.1% sodium borohydride in PBS prior to antibody incubation and/or by selecting probes and optical filters that maximize the fluorescence signal relative to the autofluorescence. Fourthly, fluorescence overlap (also termed bleed-through, crossover or crosstalk) is mainly due to the emission spectral properties of the fluorophores as they often exhibit very broad bandwidths, different, asymmetrical spectral profiles, as well as various peak emission wavelengths and number of maxima. Fluorescence overlap occurs when working with several fluorophores (multicolor labeling) and is characterized by the emission of one fluorophore in the channel (filter) of another fluorophore. Bleed-through artifacts must be minimized as they often complicate the interpretation of IF results, particularly in the case of co-localization or quantitative studies. As balancing of the fluorophores emission can only be slightly improved by the IF procedure, bleed-through can mainly be reduced at time of image acquisition by using an optimized fluorescence filter sets and/or photomultiplier detector in order to properly separate the spectral profiles of the fluorophores. In this regard, confocal microscopy is well-suited to multicolor imaging as it allows the differentiation fluorescence emission spectra of individual fluorophores by directing each signal to a particular detection channel. Moreover, confocal microscopy allows to adjust the gain, photomultiplier voltage, or laser power for the individual detection channels for the sequential acquisition (only one fluorophore at a time) of the labeling. Ideally, single-label controls must be performed to quantify the bleed-through and eventually remove it computationally. A control without secondary antibodies (background control) can be prepared to set the limits of signal gain and offset of each channel for optimal image acquisition. It can also be used for post-acquisition processing to correct image background (autofluorescence).

In conclusion, the method described provides a simple standard protocol for easy realization of immunostaining on mammary gland sections. Nevertheless, the principal steps of an IHC experiment must be optimized for each antigen/antibody couple in order to visualize specific staining and to minimize non-specific background signals. The described method also includes several basic methods for the post-treatment of most of the images obtained. Fluorescence-based immunodetection is a powerful method with a wide range of applications from the cellular localization of an antigen to diagnosis. New advances in these approaches will be achieved with the future development of new fluorophores, acquisition devices and microscopy techniques, to image previously unobserved details of biological structures and processes.

The mammary gland is an atypical mammalian exocrine organ whose main function is to produce milk to feed newborns. The development of the mammary tissue occurs mainly after birth and is characterized by a unique process in which the epithelium invades the surrounding stroma. This tissue undergoes many changes (growth, differentiation and regression), especially during the adult life, concomitantly with variations in reproductive status (Figure 1). In addition to the overall morphology of the tissue, the proportions of different cell types as well as their arrangement within the mammary gland dramatically change during development1-5.
During embryonic life, the mammary epithelium derives from mammary milk lines, which is defined by a slight thickening and stratification of the ectoderm, between the fore and hind limbs on each side of the midline around embryonic day 10.5 (E10.5) (Figure 1A). On E11.5, the milk line breaks up into individual placodes, which are symmetrically positioned along the mammary milk line at reproducible locations, and the surrounding mesenchyme starts to condense. The placodes begin to sink deeper into the dermis and the mammary mesenchyme organizes in concentric layers around the mammary bud (E12.5-E14.5). As of E15.5, the mammary epithelium, starts to proliferate and elongate to form the primary sprout that pushes through the mammary mesenchyme towards the fat pad. The primary sprout develops a hollow lumen with an opening to the skin, marked by the formation of the nipple sheath. On E18.5, the elongating duct has grown into the fat pad and has branched into a small arborized ductal system encompassed in the fat pad. Development is essentially arrested and the rudimentary mammary gland remains morphogenetically quiescent until puberty. In the male embryo, the activation of androgen receptors leads to the degeneration of the buds, which disappear by E15.5. As of E18, mammary development ceases until puberty6-9.
At birth, the mammary gland harbors a rudimentary ductal system that elongates and branches slowly (isometric growth). At the onset of puberty, spherical structures located at the tips of the ducts called the terminal end buds (TEBs), are formed of an outer layer of cap cells and a multilayered inner core of cells (body cells). These structures are highly proliferative and infiltrate the surrounding stromal tissue in response to hormonal cues. Proliferation within the TEBs results in ductal elongation, coupled with branching morphogenesis. This process leads to the establishment of a basic epithelial arborized network emanating from the nipple (Figure 1B, puberty). At ~10-12 weeks after birth, when the epithelium has invaded the whole fat pad, its expansion stops and the TEBs disappear.&#160;Ductal development then undergoes dynamic changes, i.e., successive proliferation and regression of epithelial cells according to estrous cycles10 (Figure 1B, adult).
From the onset of gestation, the mammary tissue undergoes important growth and morphological changes to prepare for lactation. The mammary epithelium extensively proliferate and differentiate, leading to a highly branched tubulo-alveolar network. Concomitantly, mammary epithelial cells (MECs) become polarized and able to synthesize and secrete milk products. MECs organize into numerous alveolar structures (acini) that are surrounded by contractile myoepithelial cells and incorporated in a stroma composed of connective and adipose tissues, blood vessels and nerve terminals (Figure 1B, pregnancy). Furthermore, the basal side of MECs is in close contact with the basement membrane (extracellular matrix), and interactions between these two entities tightly regulate both morphogenesis and secretory function of the mammary epithelium11-13.&#160;
All these processes rely on the action of various environmental cues, of which the most important are hormones14, paracrine factors and the extracellular matrix. For example, progesterone induces extensive side-branching15 and alveologenesis that, in combination with prolactin (PRL)16,17, promotes and maintains the differentiation of the alveoli. In addition to steroids and PRL18, cytokines and signaling pathways associated with development (Wnt and Notch signaling pathways) are also involved in mammary lineage commitment and development19-21. At the end of pregnancy, the luminal MECs begin to produce a protein-rich milk known as colostrum in the lumen of the alveoli. In addition, progesterone acts on the epithelial permeability and since the tight junctions are still open, colostrum is also found in the maternal blood stream.&#160;
After parturition, the mammary epithelium takes up almost all of the mammary gland volume and is highly organized (Figure 2, mammary epithelium). Milk-producing units, namely alveoli (Figure 2, alveolus), are formed by a monolayer of polarized mammary epithelial secretory cells (MESCs), with their apical plasma membrane delimiting the lumen. Alveoli arrange themselves into lobules that are grouped into lobes connected to ducts that drain milk to the outside milieu (Figure 2, lobe). Lactation occurs, i.e., MESCs start to secrete abundant amounts of milk, primarily triggered by the drop in placental hormones (mainly progesterone) (Figure 1B, lactation). Milk protein genes are activated in a defined temporal time course ranging from pregnancy to lactation9,22,23, chiefly in response to pituitary PRL released at the time of suckling. Concomitantly, contacts between MESCs and the extracellular matrix both stimulate milk protein synthesis through signals that are mediated via the interactions between cellular integrins and laminin24,25, and suppress apoptosis in MESCs26,27. These signaling pathways result in the activation of milk protein gene promoters28 through the activation of specific transcription factors29. Cell-cell contacts are also important for some aspects of differentiation including the establishment of apical polarity and the vectorial secretion of milk products. Tight junctions rapidly close after the beginning of lactation and MESCs finely orchestrate the uptake of molecules from the blood as well as the synthesis, transport and secretion of milk components, in response to the nutritional requirements of neonates. At the time of suckling, the contraction of the myoepithelial cells surrounding the alveoli occurs in response to oxytocin and leads to milk ejection through the ducts and into the nipple. Milk is a complex fluid that contains proteins (mostly caseins), sugars (mainly lactose), lipids and minerals, as well as bioactive molecules such as immunoglobulins A (IgA), growth factors and hormones. Caseins are synthesized, assembled in supramolecular structures, namely casein micelles, transported along the secretory pathway, and then released by exocytosis, i.e., the fusion of casein-containing secretory vesicles (SVs) with the apical plasma membrane of MESC (Figure 2).&#160;
Intracellular traffic relies on material exchanges between membranous compartments and involves Soluble N-ethylmaleimide-Sensitive Fusion (NSF) Attachment Protein (SNAP) Receptor (SNARE)30,31. The SNARE proteins family is subdivided in vesicular SNAREs (v-SNAREs), present in the vesicle membrane, and target SNAREs (t-SNAREs), localized on the target membranes. By zipping through their coiled-coil domains, v- and t-SNAREs assemble to form a highly stable four-helix bundle complex, referred to as the SNARE complex. This complex promotes the fusion of two opposing lipid bilayers by gradually bringing them into close proximity30,32. Afterwards, SNARE complexes are dissociated by the NSF adenosine triphosphatase and its adaptor protein SNAP and SNARE proteins are recycled back to their compartment of origin33. Interestingly, each SNARE protein predominantly resides in distinct cellular compartments and SNARE pairing may contribute to the specificity of intracellular fusion events34. Previous studies suggest that at least Synaptosomal-Associated Protein 23 (SNAP23) and Vesicle-Associated Membrane Protein 8 (VAMP8), and syntaxins (Stx) -7 and -12 play a role in casein exocytosis 35,36. These proteins have also been found in association with the lipid fraction of milk, i.e., milk fat globules (MFGs)37. The current prevailing model postulates that cytoplasmic lipid droplets (CLDs) are formed by the accumulation of neutral lipids (mainly triacylglycerols and sterol esters) and cholesterol derived from the maternal diet between the two leaflets of the endoplasmic reticulum (ER) membrane38-41. Large CLDs are formed, at least in part, by the fusion of smaller CLDs while being transported to the apical side of MESCs where they are released as MFGs (1-10 &#181;m in diameter) by budding, being enwrapped by the MESC apical plasma membrane40-42. Lactation ceases after pups are weaned and the MESCs progressively die by apoptosis, leading to the regression of the mammary tissue back to a pubertal state (Figure 1B, involution).
Immunofluorescence (IF) is a common analytical laboratory method used in almost all aspects of biology, both in research and in clinical diagnostics. IF techniques can be performed on tissue sections (immunohistochemistry, IHC) or cell (immunocytochemistry, ICC) samples. This powerful approach relies on the use of fluorescent-labeled antibodies that specifically bind (directly or indirectly) to the antigen of interest, thus allowing the visualization of its tissue distribution through fluorescence microscopy. Fluorescence signals mostly depend on the quality and concentration of the antibodies and proper handling of the specimen. A simple indirect immunofluorescence (IIF) protocol is presented to detect milk products (caseins and MFGs) and proteins involved in milk product secretion (butyrophilin (BTN1), SNARE proteins) on frozen sections of mouse mammary tissue (Figure 3). While this protocol provides a complete IHC overview, ranging from tissue collection to image post-treatment, critical and optional steps as well as some technical recommendations are also presented and discussed.

<table><tbody><tr><td>Dissection</td><td></td><td></td><td></td></tr><tr><td>Pins</td><td></td><td></td><td></td></tr><tr><td>Ethanol</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Scalpel and adapted blades</td><td></td><td></td><td></td></tr><tr><td>Ice</td><td></td><td></td><td></td></tr><tr><td>Towel paper</td><td></td><td></td><td></td></tr><tr><td>Tissue sample preparation</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>Phosphate Buffered Saline (pH7.4)</td><td>Sigma</td><td>P-3813</td><td></td></tr><tr><td>Paraformaldehyde (PFA, 32% EM grade, 100 ml)</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td>personnal protection equipment required WARNING: this product will expose you to Formaldehyde Gas, a chemical known to cause cancer</td></tr><tr><td>OCT compound/Tissue Tek</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>Sucrose (D-saccharose)</td><td>VWR</td><td>27480.294</td><td></td></tr><tr><td>Plastic molds</td><td>Dominique Dutscher</td><td>39910</td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Cryostat/sample support</td><td>Leica&amp;nbsp;</td><td>CM3050S</td><td></td></tr><tr><td>Razor blades (SEC35)</td><td>Thermo Scientific</td><td>152200</td><td></td></tr><tr><td>Slide box</td><td></td><td></td><td></td></tr><tr><td>Glass slides Superfrost/Superfrost Ultra Plus</td><td>Thermo Scientific</td><td>10143560W90/1014356190</td><td></td></tr><tr><td>Brushes</td><td></td><td></td><td></td></tr><tr><td>IHC</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>Super Pap Pen</td><td>Sigma</td><td>Z377821-1EA</td><td></td></tr><tr><td>Permanent marker (black)</td><td></td><td></td><td></td></tr><tr><td>50 mM NH&lt;sub&gt;4&lt;/sub&gt;Cl in PBS</td><td>Sigma</td><td>A-0171</td><td></td></tr><tr><td>0.1 M glycine in PBS</td><td>VWR</td><td>24403.367</td><td></td></tr><tr><td>Antigen Retrieval solution: Tris 100 mM 5% urea pH9.6</td><td></td><td></td><td></td></tr><tr><td>Heater (up to 100&amp;deg;C)</td><td></td><td></td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A7906-100G</td><td></td></tr><tr><td>Vectashield (anti-fading mounting medium) without DAPI/with DAPI</td><td>Vector Laboratories</td><td>H-1000/H-1200</td><td></td></tr><tr><td>Glass coverslips 22x50mm (microscopy grade)</td><td>VWR</td><td>CORN2980-225</td><td></td></tr><tr><td>Nail polish</td><td></td><td></td><td></td></tr><tr><td>Primary antibodies</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>Rabbit anti-mouse caseins (#7781; 1:50 dilution)</td><td>generously gifted by M.C. Neville (University of Colorado Health Sciences&lt;br/&gt; Center, USA)</td><td></td><td></td></tr><tr><td>Mouse anti-cytokeratin 8 (CK8, clone 1E8, 1:50 dilution)</td><td>Biolegend (Covance)</td><td>MMS-162P</td><td></td></tr><tr><td>Mouse anti-cytokeratin 14 (CK14, cloneLL002, 1:50 dilution)</td><td>Thermo Scientific</td><td>MS-115-P0/P1</td><td></td></tr><tr><td>Rabbit anti-butyrophilin (1:300 dilution)</td><td>generously gifted by I.H. Mather (Department of Animal and Avian Sciences University of Maryland College Park, USA)</td><td></td><td></td></tr><tr><td>Rabbit anti-Stx6 (1:50 dilution)</td><td>generously gifted S. Tooze&lt;br/&gt; (Cancer Research UK, London Research Institute, London, UK)</td><td></td><td></td></tr><tr><td>Rabbit anti-VAMP4 (1:50 dilution)</td><td>Abcam</td><td>ab3348</td><td></td></tr><tr><td>Secondary antibodies</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>Rhodamine-conjugated goat anti-rabbit IgG (H + L) (1:300 dilution)</td><td>Jackson ImmunoResearch Laboratories</td><td>111-025-003</td><td></td></tr><tr><td>Counterstains</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>Bodipy 493/503</td><td>Life Technologies (Molecular Probes)</td><td>D-3922</td><td></td></tr><tr><td>DAPI (4-6-diamidino-2-phenylindole)</td><td>Life Technologies (Molecular Probes)</td><td>D-1306</td><td></td></tr><tr><td>Observation/Image capture</td><td>&lt;span style=&quot;line-height: 20.8px;&quot;&gt;Company&lt;/span&gt;</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>conventional fluorescence microscope</td><td>Leica Leitz DMRB&lt;br/&gt; microscope</td><td></td><td>Standard filters for FITC, Rhodamine&lt;br/&gt; and DAPI emissions,&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; &amp;times;63 oil-immersion objective (NA 1.3), DP50 imaging camera (Olympus), Cell&amp;circ;F software (Olympus)</td></tr><tr><td>Laser Scanning Microscope (confocal microscopy)</td><td>Zeiss LSM&lt;br/&gt; 510 microscope</td><td></td><td>Plan-Apochromat &amp;times;63 oil-immersion objective (NA 1.4),&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; CLSM 510 software,&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Confocal facilities, MIMA2 Platform, INRA Jouy-en-Josas, France, http://mima2.jouy.inra.fr/mima2)</td></tr><tr><td>Image treatment</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>ImageJ 1.49k software</td><td></td><td></td><td>Free software</td></tr></tbody></table>

indirect immunofluorescence on frozen sections of mouse mammary gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

Watch this Scientific Journal Video about Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland at JoVE.com

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

indirect-immunofluorescence-on-frozen-sections-of-mouse-mammary-gland

Universidad San Sebastian

Research

JoVE Journal

Biology

36.8K Views.  INRA. The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Video: Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Cancer Research

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

Developmental Biology

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play a major role in mammary gland function and are the origin of most mammary tumors. Introducing genes of interest into mouse mammary epithelial cells is a critical step in evaluating gene function in epithelial cells and generating mouse mammary tumor models. This goal can be accomplished through the intraductal injection of a viral vector carrying the genes of interest into the mouse mammary ductal tree. The injected virus subsequently infects mammary epithelial cells, bringing in the genes of interest. The viral vector can be lentiviral, retroviral, adenoviral, or adenovirus-associated viral (AAV). This study demonstrates how a gene of interest is delivered into mammary epithelial cells through mouse mammary intraductal injection of a viral vector. A lentivirus carrying GFP is used to show stable expression of a delivered gene, and a retrovirus carrying Erbb2 (HER2/Neu) is used to demonstrate oncogene-induced atypical hyperplastic lesions and mammary tumors.

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

The present protocol describes intraductal injection of viral vectors via the teat to deliver genes of interest into the mammary epithelial cells.

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play ...

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has a broad spectrum of applications. Detection of antigens allows for characterization and identification of multiple cell types. Located above the kidneys and encapsulated by a layer of mesenchymal cells, the adrenal gland is an endocrine organ composed by two different tissues with different embryological origins, the mesonephric intermediate mesoderm-derived outer cortex and the neural crest-derived inner medulla. The adrenal cortex secretes steroids (i.e., mineralocorticoids, glucocorticoids, sex hormones), whereas the adrenal medulla produces catecholamines (i.e., adrenaline, noradrenaline). While conducting adrenal research, it is important to be able to distinguish unique cells with different functions. Here we provide a protocol developed in our laboratory that describes a series of sequential steps required for obtaining immunofluorescence staining to characterize the cell types of the adrenal gland. We focus first on the dissection of the mouse adrenal glands, the microscopic removal of periadrenal fat followed by the fixation, processing and paraffin embedding of the tissue. We then describe sectioning of the tissue blocks with a rotary microtome. Lastly, we detail a protocol for immunofluorescent staining of adrenal glands that we have developed to minimize both non-specific antibody binding and autofluorescence in order to achieve an optimal signal.

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...

Immunology and Infection

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in ...

Intravital Subcellular Microscopy of the Mammary Gland

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676&#160;or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...

Immunofluorescence Microscopy: Immunofluorescence Staining of Paraffin-Embedded Tissue Sections

Source: Thomas Chaffee1, Thomas S. Griffith2,3,4, and Kathryn L. Schwertfeger1,3,4 
1 Department of Lab Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455 
2 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
3 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455 
4 Center for Immunology, University of Minnesota, Minneapolis, MN 55455
Pathologic analyses of tissue sections can be used to obtain a better understanding of normal tissue structure and contribute to our understanding of mechanisms of disease. Tissue biopsies, either from patients or from experimental in vivo models, are often preserved by fixing in formalin or paraformaldehyde and embedding in paraffin wax. This allows for long-term storage and for the tissues to be sectioned. Tissues are cut into thin (5 &#181;m) sections using a microtome and the sections are adhered to glass slides. The tissues sections can be stained with antibodies, which allow for the detection of specific proteins within the tissue sections. Staining with antibodies conjugated to fluorophores (also known as fluorochromes) - compounds that emit light at specific wavelengths when excited by a laser - is known as immunofluorescence. The ability to detect proteins within a section can provide information such as cell type heterogeneity within the tissue, activation of specific signaling pathways, and expression of biomarkers. Depending on the fluorophores used and type of microscope available for analysis, multiple colors can be used, which allows for multiplexed analysis of targets.
The following protocol outlines the basic steps involved in immunofluorescence staining of paraffin embedded tissue sections. It is important to note that this protocol will not include any details on the fixation of tissue, process of paraffin embedding, or sectioning of the tissues. Once tissues have been sectioned and placed on glass slides, they are rehydrated through a series of graded ethanol (EtOH) incubations. The sections are incubated with a blocking reagent to reduce non-specific binding of antibody to the tissue section. The sections are then incubated with a primary antibody that may or may not be directly labeled with a fluorophore. If the primary antibody is not directly labeled, the sections are then incubated with a secondary antibody labeled with a fluorophore. Different antibodies may require different staining conditions, thus suggestions for optimization of antibodies are included. Following washing to remove all unbound antibody, the slides are mounted with media containing DAPI to fluorescently label the nucleus. Once the mounting media has dried, the slides can be imaged using a microscope with lasers that can detect the different fluorophores.

Immunofluorescence Staining and Microscopy

Source: Thomas Chaffee1, Thomas S. Griffith2,3,4, and Kathryn L. Schwertfeger1,3,4
1 Department of Lab Medicine and Pathology, University of Minnesota, ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Summary

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Chapters in this video

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

Intravital Subcellular Microscopy of the Mammary Gland

Immunofluorescence Microscopy: Immunofluorescence Staining of Paraffin-Embedded Tissue Sections