I.P. is funded by a Natural Sciences and Engineering Research Council of Canada grant (NSERC #418233-2012); a Fonds de Recherche du Qu&#233;bec-Sant&#233; (FRQS), a Quebec Breast Cancer Foundation career award, and a Leader Founds grant from the Canadian Foundation for Innovation grant. E.D. received a scholarship from the Fondation universitaire Armand-Frappier.

All animal protocols used in this study were approved by the University Animal Care Committee (INRS-Institut Armand-Frappier, Laval, Canada).
1. Identifying Protein Co-localization
<ol>
	<li>From tissue to microscopic slides 
	NOTE: Tissues and sections should be handled on dry ice.
	<ol>
		<li>Excise the mammary glands from an animal (for a complete description of this procedure, refer to Plante et al.)14.</li>
		<li>Embed the excised tissue in freezing/mounting medium on dry ice. Add enough medium to cover the gland. When the medium is solidified, transfer the tissues to a freezer at -80 &#176;C for later use9.</li>
		<li>Using a cryomicrotome set at &#8804; -35 &#176;C, cut the tissues into 7-10 &#956;m-thick sections and place them on microscope slides. 
		NOTE: When possible, place two sections on each slide; the left section will be used as a negative control to verify the specificity of the antibodies and the autofluorescence of the tissue, while the right side will be labeled with the antibodies.</li>
		<li>Keep the sections at -80 &#176;C for later use.</li>
	</ol>
	</li>
	<li>Co-IF staining
	<ol>
		<li>Retrieve the appropriate microscopic slides from the freezer and immediately fix the sections by immersing them in formaldehyde 4% for 15 min at room temperature (RT).</li>
		<li>Then immerse the slides in phosphate-buffered saline (PBS) at RT. Leave the slides in PBS at RT until proceeding to the next step.</li>
		<li>Circle each section of the slide using a commercially available hydrophobic barrier or a water-repellent lab pen (see the Table of Materials). Be careful not to touch the tissue. Immediately add drops of PBS to the tissue and place the slide in a humid histology chamber for the remainder of the procedure. 
		NOTE: The tissue sections must remain moisturized. Alternatively, use a box with a lid and place wet paper towels on the bottom.</li>
		<li>Block each tissue section with 100-200 &#181;L of 3% bovine serum albumin (BSA)-Tris-buffered saline (TBS)-0.1% polysorbate 20 (see the Table of Materials) for 30 min at RT. While the samples are blocking, prepare the primary and secondary antibody solutions by diluting the antibodies in TBS-0.1% polysorbate 20. 
		NOTE: The required concentration for the antibody is provided by the manufacturer; see the Table of Materials and Figures 1 and 2 for examples, as well as Dianati et al.9. Although it is not necessary to work in the dark when using most fluorophore-conjugated antibodies, avoid exposing the antibody solutions or stained tissues to intense, bright light.</li>
		<li>Remove the blocking solution by aspiration and incubate the sections in 100-200 &#181;L of the diluted primary antibody for 60 min at RT. Alternatively, incubate with the primary antibody overnight at 4 &#176;C.</li>
		<li>Remove the primary antibody solution by aspiration and wash the sections with 250-500 &#181;L of TBS-0.1% polysorbate 20 for 5 min. Remove the wash solution by aspiration and repeat the wash twice.</li>
		<li>Remove the wash solution by aspiration and incubate the sections with 100-200 &#181;L of the appropriate fluorophore-conjugated secondary antibody for 60 min at RT.</li>
		<li>Remove the secondary antibody solution by aspiration and wash the sections with 250-500 &#181;L of TBS-0.1% polysorbate 20 for 5 min. Remove the wash solution and repeat twice.</li>
		<li>Repeat step 2.5-2.8 using the appropriate combination of primary and secondary antibodies for the subsequent protein(s) of interest.</li>
		<li>Remove the wash solution by aspiration and perform the nuclei staining by incubating the section with 100-200 &#181;L of 1 mg/mL 4&#39;,6-diamidino-2-phenylindole (DAPI) in TBS-0.1% polysorbate 20 for 5 min at RT.</li>
		<li>Remove the DAPI solution by aspiration and mount the slides using a water-soluble, non-fluorescing mounting medium (see the Table of Materials) and coverslips. Proceed one slide at a time. 
		NOTE: Incubating the nucleus stain for more than 5 min will not change the intensity of the staining. Alternatively, remove the DAPI solution on all slides and incubate the tissues in PBS while mounting the slides.</li>
		<li>Place the slides flat in a 4 &#176;C refrigerator for at least 8 h. Proceed to the fluorescence microscopic imaging (see Figures 1 and 2).</li>
	</ol>
	</li>
	<li>Microscopic imaging
	<ol>
		<li>Visualize the fluorophore-conjugated secondary antibodies using a confocal microscope equipped with the various lasers required to excite the fluorophores at their specific wavelengths. 
		Note: To be able to visualize ducts and alveoli, a 40X objective with a numerical aperture of 0.95 is suggested. An example of the specific settings is provided in Figure 1.</li>
		<li>Verify the localization of each protein individually by scanning the image one wavelength at the time. 
		Note: At this stage, it is important to critically analyze the localization of the junctional proteins. To be able to form junctional nexuses, these proteins must be localized at the plasma membrane.</li>
		<li>Determine the co-localization of proteins by merging the images scanned with the lasers at the different wavelengths. 
		Note: Protein co-localization can be visualized by the change of color resulting from the emission of two or more fluorophores at the same location and can be measured using the appropriate software (Figures 1 and 2; also see Reference 9).</li>
	</ol>
	</li>
</ol>
2. Studying PPIs
NOTE: Abdominal mammary glands should be used to study PPIs, as thoracic glands are in close association with the pectoral muscles. Excise the mammary glands (for a complete description of this procedure, refer to Plante et al.)14 and keep them at -80 &#176;C for later use.
<ol>
	<li>Lysate preparation
	<ol>
		<li>Place weighing paper and 2 mL microcentrifuge tubes on dry ice to pre-cool them before proceeding with the next steps.</li>
		<li>Take the mammary gland tissue from -80 &#176;C and keep them on dry ice.</li>
		<li>Weigh the tissues on the pre-chilled weighing papers and then transfer the tissue to 2 mL microcentrifuge tubes (handle on dry ice). Use between 50 and 100 mg of tissue per sample. Keep the tissue on dry ice until step 2.1.5.</li>
		<li>Prepare the required amount of triple detergent lysis buffer supplemented with NaF, NaVO3, and protease/phosphatase inhibitor, as indicated in the Table of Materials, using the following formulas. Mice: required buffer (&#181;L) = mouse tissue weight (mg) x 3; Rat: required buffer (&#181;L) = rat tissue weight (mg) x 5.</li>
		<li>Add the required amount of ice-cold lysis buffer (calculated in step 2.1.4) to the 2 mL tube containing the tissue. 
		NOTE: In steps 2.1.5-2.1.6, proceed with one single tube at a time.</li>
		<li>Homogenize the tissue for 30-40 s using continuous homogenization on a tissue grinder; always keep the tube on ice. Adjust the tissue homogenizer to medium speed and gently move the grinder up and down inside the tube.</li>
		<li>Repeat steps 1.6 and 1.7 with the other tubes.</li>
		<li>Incubate the lysates on ice for 10-30 min.</li>
		<li>Centrifuge the tubes at 170 x g for 10 min at 4 &#176;C.</li>
		<li>Meanwhile, identify 6-10 microcentrifuge tubes (0.6 mL) for each sample and keep them on ice.</li>
		<li>Once the centrifugation is done, check the tubes. Ensure that they contain a top layer of fat, clear, yellow-to-pink lysates (depending on the stage of development) and a pellet.</li>
		<li>Create a hole in the lipid layer using a 200-&#181;L pipette tip to access the liquid phase. Change the tip and collect the lysate without disturbing the pellet or aspirating the lipid layer. Aliquot the lysate in pre-labeled tubes on ice (step 2.1.11) and store them at -80 &#176;C.</li>
		<li>Use an aliquot to quantify the protein concentration using an appropriate commercially available kit (see the Table of Materials).</li>
	</ol>
	</li>
	<li>Indirect immunoprecipitation
	<ol>
		<li>On ice, thaw two aliquots of the total mammary gland lysates prepared previously. 
		NOTE: One aliquot will be used for the IP of the target protein, while the other will serve as the negative control.</li>
		<li>Collect 500-1,000 &#181;g of the lysate and dilute it in PBS to reach a final volume of 200 &#181;L in each 1.5 mL tube. 
		NOTE: The amount of lysate to be used depends on the abundance of the protein of interest and the efficiency of the antibody (see Figure 3 for an example, as well as the Table of Materials). To optimize for each target, different amounts of lysate (i.e., 500, 750, and 1,000 &#181;g) and antibody (i.e., 5, 10, and 20 &#181;g) should be used. Proceed with the following steps (2.2.3-2.3.7.4).</li>
		<li>Add the antibody against the antigen of interest to the first tube of lysate and keep it on ice. 
		NOTE: The required amount is usually suggested on the instruction sheet provided by each company (see the Table of Materials).</li>
		<li>In the second tube, prepare a negative control by adding the same concentration of isotype IgG control as the antibody used in step 2.2.3.</li>
		<li>Incubate the tubes overnight at 4 &#176;C on a tube roller-mixer at low speed.</li>
		<li>The following day, add 50 &#181;L of magnetic beads to new 1.5 mL tubes for pre-washing.
		<ol>
			<li>Select either Protein A or Protein G magnetic beads based on the relative affinity to the antibody.</li>
			<li>It is important to avoid using aggregated beads; gently mix the bead suspension until it is uniformly re-suspended before adding it to the tubes.</li>
		</ol>
		</li>
		<li>Place the tubes containing the beads on the magnetic stand and allow the beads to migrate to the magnet. Remove the storage buffer from the beads using a 200 &#181;L pipette.</li>
		<li>Wash the beads by adding 500 &#181;L of PBS-0.1% polysorbate 20 and vortex the tubes vigorously for 10 s.</li>
		<li>Put the tubes back on to the magnetic stand and allow the beads to migrate to the magnet.</li>
		<li>Remove the excess wash buffer by pipetting with a 200 &#181;L pipette.</li>
		<li>Add the reaction complex (lysate-antibody) from step 2.2.5 to the beads and incubate for 90 min at RT on the roller mixer.</li>
		<li>Place the tubes on the magnetic stand and allow the beads to migrate to the magnet. Using a 200 &#181;L pipette, aspirate and discard the lysate and place the tubes on ice.</li>
		<li>Wash the beads by adding 500 &#181;L of PBS, placing the tubes on the magnetic stand, and removing the liquid using a 200 &#181;L pipette. Repeat this wash step. During the wash steps, avoid vortexing and keep the samples on ice.</li>
		<li>Wash the beads once with PBS-0.1% polysorbate 20 without vortexing and discard the last wash buffer using a 200 &#181;L pipette tip.</li>
		<li>To elute, add 20 &#181;L of 0.2 M acidic glycine (pH = 2.5) to the tubes and shake them for 7 min on the roller mixer.</li>
		<li>Centrifuge at high speed for a few seconds (quick spin) and collect the supernatant in a fresh ice-cold tube.</li>
		<li>Repeat steps 2.2.14 and 2.2.15 for each tube. 
		NOTE: The final volume will be 40 &#181;L.</li>
		<li>Add 10 &#181;L of 4x Laemmli buffer to the 40-&#181;L eluted sample from step 2.2.16. 
		NOTE: The color will turn yellow due to the acidic pH.</li>
		<li>Immediately add 1 M Tris (pH = 8), one drop at a time, to the eluted sample from step 2.2.18 until its color turns blue. Proceed to the next tubes.</li>
		<li>Boil the samples from step 2.2.18 at 70-90 &#176;C for 10 min. Proceed immediately to gel electrophoresis. Alternatively, transfer the samples to a freezer at -80 &#176;C until loading.</li>
	</ol>
	</li>
	<li>Downstream application: gel electrophoresis followed by Western blot
	<ol>
		<li>Prepare separating and stacking SDS-PAGE acrylamide gels (1.5 mm thickness) following standard procedures15. 
		NOTE: The choice of gel (8-15% acrylamide, gradient: see the Table of Materials) should be determined based on the molecular size of the protein to be precipitated and of the potential binding partners to be analyzed. These proteins must be resolved from each other to allow for proper immunodetection.</li>
		<li>Thaw the immunoprecipitation (IP)-eluted samples (step 2.2.20) on ice.</li>
		<li>Prepare protein lysates from the same samples (used for the IP procedure above). Use 50 &#181;g of total lysate and add 4X Laemmli sample buffer. Boil the samples at 70-90 &#176;C for 5 min and place on ice until loading. 
		NOTE: These samples will be loaded beside the eluted IP sample to demonstrate the presence of precipitated proteins in the total lysate.</li>
		<li>Load the prepared lysates from step 2.3.3 and the precipitated samples from step 2.2.20 side-by-side in an acrylamide gel and run them in running buffer (10x running buffer: 30.3 g of Tris, 144.1 g of glycine, and 10 g of SDS in 1 L of distilled water) at 100 V for approximatively 95 min, or until the edge of the migrating proteins reaches the bottom of the gel.</li>
		<li>Transfer the gels to a nitrocellulose or PVDF membrane using a standard protocol9,15.</li>
		<li>Block the membrane for 1 h on a rocker on low speed in 5% dry milk-TTBS (20 mM Tris, 500 mM NaCl, and 0.05% polysorbate 20).</li>
		<li>Identify whether the precipitation was successful.
		<ol>
			<li>Probe the membrane using the first antibody against the precipitated protein, diluted in 5% dry-milk-TTBS at the concentration recommended by the manufacturer, overnight at 4 &#176;C on a rocking platform with slow agitation. 
			NOTE: See the Table of Materials for recommendations.</li>
			<li>The following day, wash the membrane 3 times for 5 min each with TTBS on a rocking platform with high agitation.</li>
			<li>Incubate the membrane in the appropriate secondary antibody conjugated with horseradish peroxidase (HRP), diluted in TTBS, for 1 h at RT on a rocking platform with slow agitation. 
			NOTE: Alternatively, a secondary antibody conjugated with a fluorochrome can be used if an appropriate apparatus to detect the signal is available.</li>
			<li>Perform 3 to 6 washes, each for 5 min, with TTBS on a rocking platform with high agitation. Analyze the signal of the secondary antibody by incubating the membrane with a commercially available luminol solution (see the Table of Materials) and follow the manufacturer instructions. Detect the signal using a chemiluminescence imaging system (see the Table of Materials). 
			NOTE: For a detailed protocol on Western blot analysis, see Reference 16.</li>
		</ol>
		</li>
		<li>To identify interacting proteins, perform steps 2.3.7.1-2.3.7.4 using the appropriate antibodies on the same blot. 
		NOTE: If proteins are interacting, the binding partners will be co-immunoprecipitated with the target protein and will thus be detectable by Western blotting. Step 2.3.8 can be repeated with more antibodies to determine whether other proteins reside in the same proteins complex, as long as the molecular weights of the proteins differ enough to be well-separated on the gel and membrane.</li>
		<li>To confirm that the identified binding partners are not artifacts, reciprocal IP should be performed. 
		NOTE: This is performed by repeating steps 2.2.1-2.2.20 with the same lysate but precipitating one of the binding partners identified in step 3.8. Then, steps 3.1-3.8 are repeated using the primary antibody against the first protein of interest.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to declare.

Cell-cell interactions via junctions are required for the proper function and development of many organs, such as the mammary gland. Studies have shown that junctional proteins can regulate the function and stability of one another and activate signal transduction by tethering each other at the cell membrane10. The protocols presented in the current manuscript have provided interesting findings about junctional protein differential expression, localization, and interaction during normal murine gla...

Mammary gland growth and development occurs mainly after birth. This organ constantly remodels itself throughout the reproductive life of a mammal1. The adult mammary gland epithelium is comprised of an inner layer of luminal epithelial cells and an outer layer of basal cells, mainly composed of myoepithelial cells, surrounded by a basement membrane2. For a good review on mammary gland structure and development, the reader can refer to Sternlicht1. Cell-cell interactions via gap (GJ), tight (TJ), and adherens (AJ) junctions are necessary for the normal development and function of the gland1,3,4,5,6. The main components of these junctions in the murine mammary gland are Cx26, Cx30, Cx32, and Cx43 (GJ); Claudin-1, -3, -4, and -7 and ZO-1 (TJ); and E-cadherin, P-cadherin, and &#946;-catenin (AJ)7,8. The levels of expression of these different junctional proteins vary in a stage-dependent manner during mammary gland development, suggesting differential cell-cell interaction requirements9. GJ, TJ, and AJ are linked structurally and functionally and tether other structural or regulatory proteins to the neighboring sites of adjacent cells, thus creating a junctional nexus10. The composition of the junctional nexus can impact bridging with the underlying cytoskeleton, as well as nexus permeability and stability, and can consequently influence the function of the gland8,9,10,11. The components of intercellular junctions residing in junctional nexuses or interacting with one another at different developmental stages of mammary gland development were analyzed recently using co-immunofluorescence (co-IF) and co-immunoprecipitation (co-IP)9. While other techniques allow for the evaluation of the functional association between proteins, these methods are not presented in this manuscript.
As proteins merely act alone to function, studying protein-protein interactions (PPIs), such as signal transductions and biochemical cascades, is essential to many researchers and can provide significant information about the function of proteins. Co-IF and microscopic analysis help to evaluate a few proteins that share the same subcellular space. However, the number of targets is limited by the antibodies, which must be raised in different animals, and by the access to a confocal microscope equipped with different wavelength lasers and a spectral detector for multiplexing. Co-IP confirms or reveals high-affinity physical interactions between two or more proteins residing within a protein complex. Despite the development of novel techniques, such as fluorescence resonance energy transfer (FRET)12 and proximity ligation assay (PLA)13, which can simultaneously detect the localization and interactions of proteins, co-IP remains an appropriate and affordable technique to study interactions between endogenous proteins.
The step-by-step method described in this manuscript will facilitate the study of protein localization and PPIs and point out pitfalls to avoid when studying endogenous PPIs in the mammary glands. The methodology starts with the presentation of the different preservation procedures for the tissues required for each technique. Part 1 presents how to study protein co-localization in three steps: i) the sectioning of mammary glands, ii) the double- or triple-labeling of different proteins using the co-IF technique, and iii) the imaging of protein localization. Part 2 shows how to precipitate an endogenous protein and identify its interacting proteins in three steps: i) lysate preparation, ii) indirect protein immunoprecipitation, and iii) binding partner identification by SDS-PAGE and Western blot. Each step of this protocol is optimized for rodent mammary gland tissues and generates high-quality, specific, and reproducible results. This protocol can also be used as a starting point for PPI studies in other tissues or cell lines.

<table><tbody><tr><td>Mice strain and stage</td><td>St. Constant, Quebec, Canada</td><td></td><td>C57BL/6 Femals; pregnancy day 18 (P18) and lactation day 14 (L14), Charles River Canada&amp;nbsp;</td></tr><tr><td>PBS 10x (stock)</td><td></td><td></td><td>1) Dissolve 80 g NaCl (F.W.: 58.44), 2 g KCl (F.W. 74.55), 26.8 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;#183;7H&lt;sub&gt;2&lt;/sub&gt;O (F.W. 268.07) and 2.4 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (F.W.:136.09) in 800 mL distilled water;&lt;br/&gt; 2) Adjust the PH to 7.4;&lt;br/&gt; 3) Add water to reach to the 1 L final volume.</td></tr><tr><td>TBS 10x (stock)</td><td></td><td></td><td>1) Dissolve 60.5 g TRIS, 87.6 g NaCl in 800 mL distilled water;&lt;br/&gt;2) Adjust the pH to 7.5;&lt;br /&gt;3) Add water to reach to the 1 L final volume.</td></tr><tr><td colspan="4">&lt;strong&gt;Part 1: Immunofluorescence&lt;/strong&gt;</td></tr><tr><td>Freezing media</td><td>VWR International, Ville Mont-Royal, QC, Canada</td><td>95067-840</td><td>VMR frozen sections compound&amp;nbsp;</td></tr><tr><td>Microtome</td><td>Mississauga, ON, Canada</td><td>956640</td><td>Microm HM525, Thermo fisher scientific HM525 NX Cryostat 115 V 60 Hz</td></tr><tr><td>Blades</td><td>C.L. Sturkey, Inc. Les Produits&amp;nbsp;Scientifiques&amp;nbsp;ESBE&amp;nbsp;St-Laurent, QC, Canada</td><td>BLM1001C</td><td>High profile gold coated blades</td></tr><tr><td>Pap pen</td><td>Cedarlane, Burlington, ON, Canada</td><td>8899</td><td>Super PAP Pen, Thermo fisher scientific</td></tr><tr><td>Microscopic slides</td><td>Fisher Scientific, Burlington, ON, Canada</td><td>12-550-15</td><td>Fisherbrand&amp;nbsp;Superfrost Plus Microscope Slides</td></tr><tr><td>Formaldehyde</td><td>BioShop Canada Inc, Burlington, ON, Canada</td><td>FOR201.1</td><td>Forlmadehyde</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Santa Cruz Biotechnology, Inc, California, USA</td><td></td><td></td></tr><tr><td>Blocking solution</td><td></td><td></td><td>3% BSA in TBS</td></tr><tr><td>Wash solution</td><td></td><td></td><td>TBS-Tween 20 0.1%</td></tr><tr><td>Polysorbate 20</td><td>Oakville, ON, Canada</td><td>P 9416</td><td>Tween 20, Sigma-Aldrich</td></tr><tr><td>Mounting media</td><td>Cedarlane, Burlington, ON</td><td>17984-25(EM)</td><td>Fluoromount-G</td></tr><tr><td>First &amp;amp; secondary antibodies</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>E-Cadherin (4A2) Mouse mAb (#14472s) 1/50 (Cell Signaling) with anti-mouse IgG Fab2 Alexa Fluor 555 (#4409s), Cell Signaling&amp;nbsp;</td></tr><tr><td>First &amp;amp; secondary antibodies&amp;nbsp;</td><td>Life technologies, Waltham, MA, USA &amp;amp; Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>Claudin-7 (#34-9100) 1/100 (Life Technologies) with anti-rabbit IgG Fab2 Alexa Fluor 488 (#4412s) (Cell Signaling)&amp;nbsp;</td></tr><tr><td>First &amp;amp; secondary antibodies&amp;nbsp;</td><td>Santa Cruz Biotechnology, Inc, California, USA; Fischer Scientific, Burlington, ON, Canada&amp;nbsp;</td><td>See Comments</td><td>&amp;beta;-Catenin Antibody (C-18): sc-1496 (SANTA CRUZ) with anti-Goat IgG (H+L) Alexa Fluor 568 (#A11057), Molecular Probe (Fisher Scientific)</td></tr><tr><td>First &amp;amp; secondary antibodies&amp;nbsp;</td><td>Life technologies, Waltham, MA, USA &amp;amp; Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>Connexin26&amp;nbsp; (#33-5800) 1/75 (Life Technologies) with anti-mouse IgG Fab2 Alexa Fluor 647 (#4410s)&amp;nbsp;</td></tr><tr><td>Nuclei stain</td><td>Fisher Scientific, Burlington, ON, Canada</td><td>D1306</td><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride) 1/1,000 in PBS</td></tr><tr><td>Fluorescent microscope</td><td>Nikon Canada, Mississauga, On, Canada</td><td></td><td>Nikon A1R+ confocal microscopic laser equipped with a spectral detector&amp;nbsp;</td></tr><tr><td>Software of IF images analysis</td><td>Nikon Canada, Mississauga, On, Canada</td><td></td><td>NIS-elements software (version 4)</td></tr><tr><td colspan="4">&lt;strong&gt;Part 2: Immunoprecipitation&lt;/strong&gt;</td></tr><tr><td>Triple-detergent Lysis buffer (100&amp;nbsp;mL) pH=8.0</td><td></td><td></td><td>1) Mix 50 mM TRIS (F.W.: 121.14), 150&amp;nbsp;mM NaCl (F.W.: 58.44), 0.02% Sodium Azide, 0.1% SDS, 1% NONIdET P40, 0.5% Sodium Deoxycholate in 80 mL distilled H&lt;sub&gt;2&lt;/sub&gt;O.&lt;br/&gt; 2) Adjust the pH to 8.0 with HCl 6&amp;nbsp;N (~0.5&amp;nbsp;mL).&lt;br/&gt; 3) Adjust the volume to 100&amp;nbsp;mL. Keep it in fridge.&lt;br/&gt; At the day of protein extraction, use 1/100 NaVo&lt;sub&gt;3&lt;/sub&gt;, 1/100 protease/phosphatase inhibitor and 1/25 NAF in calculated amount of Triple detergent lysis buffer:&lt;br/&gt; Sodium Fluoride (stock) solution 1.25&amp;nbsp;M (F.W.: 41.98), Sodium Orthovanadate (stock) Solution 1&amp;nbsp;M (F.W.: 183.9)</td></tr><tr><td>Protease/phosphatase inhibitor</td><td>Fisher Scientific, Burlington, ON</td><td>78441</td><td>Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100x)</td></tr><tr><td>Protein dosage</td><td>Thermo Scientific, Rockford, Illinois, USA</td><td>23225</td><td>Pierce BCA protein assay kit&amp;nbsp;</td></tr><tr><td>Tissue grinder</td><td>Fisher Scientific, Burlington, ON</td><td>FTH-115</td><td>Power 125, Model FTH-115</td></tr><tr><td>Magnetic beads and stand</td><td>Millipore, Etobicoke, ON, Canada</td><td></td><td>PureProteome Protein G Magnetic Bead System (LSKMAGG02)</td></tr><tr><td>Wash solution for IP</td><td></td><td></td><td>PBS or PBS-Tween20 0.1% depending to the step</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>IgG Rabbit (rabbit (DA1E) mAb IgG Isotype control (#3900s) (Cell Signaling) 0.5 &amp;micro;L/200 &amp;micro;L</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>IgG Mouse mouse (G3A1) mAb IgG Isotype control (#5415s) (Cell Signaling) 0.5 &amp;micro;L/200 &amp;micro;L</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Sigma-Aldrich, Oakville, ON, Canada</td><td>See Comments</td><td>Connexin43 (#C6219) (Sigma-Aldrich) 4&amp;nbsp;&amp;micro;L/200&amp;nbsp;&amp;micro;L</td></tr><tr><td>Primary antibodies for immunoprecipitation</td><td>Cell Signaling, Beverly, MA, USA</td><td>See Comments</td><td>E-cadherin (4A2) Mouse mAb (#14472s) (Cell Signaling) 1 &amp;micro;L/200 &amp;micro;L</td></tr><tr><td>Laemmli buffer&amp;nbsp;</td><td>BIO-RAD, Mississauga, Ontario, Canada</td><td>1610747</td><td>4x Laemmli Sample Buffer (Add &amp;beta;-mercaptoethanol following manufacturer recommendation)</td></tr><tr><td>Acidic glycine&amp;nbsp;</td><td>Fisher Scientific, Burlington, ON</td><td>PB381-5</td><td>0.2 M glycine; adjust pH=2.5 with HCl&amp;nbsp;</td></tr><tr><td>Tris&amp;nbsp;</td><td>Fisher Scientific, Burlington, ON</td><td>BP152-1</td><td>1 M (pH=8)&amp;nbsp;</td></tr><tr><td>SDS-PAGE acrylamide gels&amp;nbsp;</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1610180 -5</td><td>TGX Stain-Free FastCast Acrylamide Solutionss (7.8%, 10%, 12%)</td></tr><tr><td>Running buffer 10x&amp;nbsp;</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1704272</td><td>Tris 30.3&amp;nbsp;g/glycine 144.1&amp;nbsp;g /SDS 10&amp;nbsp;g in 1 L distilled water</td></tr><tr><td>Membranes</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1704272</td><td>PVDF membranes, Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td></tr><tr><td>Transfer method</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1704155</td><td>Trans-Blot Turbo Transfer System</td></tr><tr><td>Dry Milk</td><td>Smucker Food of Canada Co, Markham, ON, Canada</td><td></td><td>Fat Free Instant Skim Milk Powder, Carnation</td></tr><tr><td>Blocking solution for blots</td><td></td><td></td><td>5% dry milk in TBS-Tween 20 0.1%</td></tr><tr><td>Washing solutions for blots</td><td></td><td></td><td>TBS-Tween 20 0.1%</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Sigma-Aldrich, Oakville, Ontario &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>Connexin43 (#C6219) (Sigma-Aldrich) 1/2,500 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Cell Signaling, Beverly, MA, USA &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>E-cadherin (24E10) rabbit mAb 1/1,000 (#3195s) (Cell Signaling) 1/1,000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Life technologies, Waltham, MA, USA &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>Claudin-7 (#34-9100) (Life technologies) 1/1,000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Primary and secondary antibodies for blots (10&amp;nbsp;mL)</td><td>Life technologies, Waltham, MA, USA &amp;amp; Abcam, Toronto, ON, Canada</td><td>See Comments</td><td>Claudin3 (#34-1700) (Life technologies) 1/1,000 with HRP-conjugated Veriblot for IP secondary antibody (ab131366) 1/5,000 (Abcam, Toronto, ON, Canada)</td></tr><tr><td>Luminol solution for signal detection on blots</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1705061</td><td>Clarity Western ECL Blotting Substrate</td></tr><tr><td>Imaging blots</td><td>BIO-RAD, Mississauga, ON, Canada</td><td>1708280</td><td>ChemiDoc MP imaging system</td></tr><tr><td>Analayzing blots</td><td>BIO-RAD, Mississauga, ON, Canada</td><td></td><td>ImageLab 5.2 software&amp;nbsp;</td></tr></tbody></table>

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.

To determine whether GJ, AJ, and TJ components can interact together in the mammary gland, co-IF assays were first performed. Cx26, a GJ protein, and &#946;-Catenin, an AJ protein, were probed with specific antibodies and revealed using fluorophore-conjugated mouse-647 (green, pseudocolor) and goat-568 (red) antibodies, respectively (Figure 1B and C). Data showed that they co-localize at the cell membrane of epithelial cells in the mice mammary gland on l...

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

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 ...

analysis-protein-protein-interactions-co-localization-between

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

10.0K Views.  INRS, Institut Armand-Frappier. 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. 

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

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Cancer Research

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Bioengineering

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Biology

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

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.

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 ...

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 ...