The research in this report was funded by a target grant from the NIGMS (1P20GM103653) awarded to DK.

The following protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Delaware. Male Sprague Dawley rats approximately 55&#8211;75 days old were used for this protocol.
1. Brain Extraction and Tissue Preparation
<ol>
	<li>Anesthetize rat with isoflurane in anesthesia induction chamber until rat no longer exhibits a response to foot pinch.</li>
	<li>Sacrifice rats via rapid decapitation using a guillotine.</li>
	<li>Cut skin on skull posterior to anterior and clear from top of skull.</li>
	<li>Use rongeurs to carefully remove the back part of skull, and then using small dissecting scissors cut down midline of skull, pushing upward against the top of skull at all times to avoid damaging the brain tissue.</li>
	<li>Use rongeurs to peel away right and left half of skull to expose the brain.</li>
	<li>Use a small spatula to scoop under brain and sever nerves, carefully elevate brain and freeze brains in isopentane chilled on dry ice (at least -20 &#176;C). After this, store brains in a -80 &#176;C freezer until slicing.</li>
	<li>Slice brains in regions of interest at 30&#8211;50 &#181;m in a cryostat maintained between -9 &#176;C and -12 &#176;C. Directly mount slices onto glass slides and store in a -80 &#176;C freezer until time of immunocytochemistry assay. 
	NOTE: In this protocol brain regions of interest were the hippocampus and the amygdala.</li>
</ol>
2. Single Immunohistochemical Reaction
NOTE: For double immunohistochemical reaction, the protocol is the same as the single immunohistochemical reaction except this reaction has two primary antibodies of different hosts (e.g., rabbit and mouse) and two secondary antibodies for the corresponding primaries should be from a single host (e.g., goat antirabbit and goat antimouse). The secondary antibodies also have to be from two different spectra that are available in high-resolution scanners. For example, one secondary antibody with an emission spectrum peak at 680 nm and one secondary antibody 800CW (emission spectrum peak at 780 nm).
<ol>
	<li>Remove glass slides from the freezer and allow to equilibrate to room temperature for 30 min.</li>
	<li>Under a fume hood, fix brain tissue in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) for 1&#8722;2 h at room temperature.</li>
	<li>Rinse slides in 0.1 M tris buffered saline (TBS) three times for 10 min each. Permeabilize cell membranes by incubating slides in a mild detergent (e.g., 0.01% detergent) for 30&#8722;60 min.</li>
	<li>Rinse slides in TBS three times for 15 min each.</li>
	<li>Dilute primary antibody for protein of interest in PBS in the correct concentration. For example, to detect the immediate early gene c-Fos, prepare a rabbit primary anti c-Fos antibody in a concentration of 1:500.</li>
	<li>Pipette primary antibody solution directly onto the brain tissue (approximately 200 &#181;L per 3 inch x 1 inch slide).</li>
	<li>Use coverslips to incubate the brain tissue on glass slides in primary antibody dilution for either 1&#8722;2 h at room temperature or overnight (~17 h) at 4 &#176;C.</li>
	<li>Remove coverslips and wash slides in TBS that has a small amount of detergent added to it (e.g., 0.01% detergent, &#34;TBS-T&#34;) four times for 15 min each.</li>
	<li>Use coverslips to incubate brain sections in a secondary antibody at room temperature for 2 h. 
	NOTE: The secondary antibody has to be to the correct dilution and in a diluent containing TBS, detergent, and 1.5% of host serum. For example, a goat secondary antibody in a dilution of 1:2,000 would contain 1.5% goat serum and 0.05% detergent.</li>
	<li>Rinse slides in TBS-T four times for 20 min each, and then in TBS four times for 20 min each.</li>
	<li>Dry slides at room temperature in the dark overnight. When slides are dry, they are ready for imaging.</li>
</ol>
3. Imaging
<ol>
	<li>Place slides onto the near-infrared scanning interface with the tissue facing down. Either image one glass slide or multiple slides at a time using a selection tool.</li>
	<li>Image slides using the highest quality setting with an offset of 0 nm and a resolution of 21 &#181;m. The scanning will typically take 13&#8722;19 h depending on the scanning equipment used.</li>
	<li>Import images into the image analysis software (e.g., ImageStudio) to view and mark for semi-quantitative protein analysis.</li>
</ol>
4. Protein Expression Analysis
<ol>
	<li>Open the image analysis software and select the Work Area into which the image was scanned.</li>
	<li>Open the scanned image in the image analysis software to view the scan, and adjust which wavelengths are viewed, as well as the contrast, brightness, and magnification shown without altering the raw image or the total quantified emission.</li>
	<li>Identify the key regions for quantification and select the Analysis tab along the top of the page, then select Draw Rectangle (or Draw Ellipse/ Draw Freehand) to draw a rectangle over the area that will be quantified.</li>
	<li>To view the size of the rectangle, select Shapes along the bottom left of the screen, then select Columns along bottom right. Add Height and Width columns to identify the shape size. 
	NOTE: It is important to control for shape size when comparing quantification. It is recommended to use identical shapes placed within the desired quantification location, in order to get an accurate sampling of the emissions for that region.</li>
	<li>Then name the shape and repeat. Once all regions are sampled the data available from the columns tab can be aggregated and analyzed.</li>
</ol>

<ol>
	<li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Neural Science. , (2013).</li><li>Byrne, J. H., Roberts, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> From Molecules to Networks: An Introduction to Cellular and Molecular Neuroscience. , (2009).</li><li>Salami, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+D2/3+binding+potential+modulates+neural+signatures+of+working+memory+in+a+load-dependent+fashion.">Dopamine D2/3 binding potential modulates neural signatures of working memory in a load-dependent fashion.</a> Journal of Neuroscience. , (2018).</li><li>Merali, Z., Khan, S., Michaud, D. S., Shippy, S. A., Anisman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+amygdaloid+corticotropin-releasing+hormone+(CRH)+mediate+anxiety-like+behaviors?+Dissociation+of+anxiogenic+effects+and+CRH+release.">Does amygdaloid corticotropin-releasing hormone (CRH) mediate anxiety-like behaviors? Dissociation of anxiogenic effects and CRH release.</a> European Journal of Neuroscience. 20 (1), 229-239 (2004).</li><li>English, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dataset+of+mouse+hippocampus+profiled+by+LC-MS/MS+for+label-free+quantitation.">Dataset of mouse hippocampus profiled by LC-MS/MS for label-free quantitation.</a> Data in Brief. 7, 341-343 (2016).</li><li>David, M. A., Tayebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+protein+aggregates+in+brain+and+cerebrospinal+fluid+derived+from+multiple+sclerosis+patients.">Detection of protein aggregates in brain and cerebrospinal fluid derived from multiple sclerosis patients.</a> Frontiers in Neurology. 5, 251 (2014).</li><li>Oliver, C., Jamur, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+methods+and+protocols.">Immunocytochemical methods and protocols.</a> Methods in Molecular Biology. , (2010).</li><li>Spitzer, N., Sammons, G. S., Price, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autofluorescent+cells+in+rat+brain+can+be+convincing+impostors+in+green+fluorescent+reporter+studies.">Autofluorescent cells in rat brain can be convincing impostors in green fluorescent reporter studies.</a> Journal of Neuroscience Methods. 197 (1), 48-55 (2011).</li><li>Knox, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+c-Jun+to+identify+fear+extinction+learning-specific+patterns+of+neural+activity+that+are+affected+by+single+prolonged+stress.">Using c-Jun to identify fear extinction learning-specific patterns of neural activity that are affected by single prolonged stress.</a> Behavioural Brain Researach. 341, 189-197 (2018).</li><li>Knox, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuits+via+which+single+prolonged+stress+exposure+leads+to+fear+extinction+retention+deficits.">Neural circuits via which single prolonged stress exposure leads to fear extinction retention deficits.</a> Learning &amp; Memory. 23 (12), 689-698 (2016).</li><li>Malinow, R., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+trafficking+and+synaptic+plasticity.">AMPA receptor trafficking and synaptic plasticity.</a> Annual Review of Neuroscience. 25, 103-126 (2002).</li><li>Nabavi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+memory+with+LTD+and+LTP.">Engineering a memory with LTD and LTP.</a> Nature. 511 (7509), 348-352 (2014).</li><li>Kimmelmann-Shultz, B., Mohammadmirzaei, N., Della Valle, R., Knox, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+high+resolution+near+infrared+imaging+to+measure+fear-learning+induced+changes+in+AMPA/NMDA+ratios+throughout+the+fear+circuit.">Using high resolution near infrared imaging to measure fear-learning induced changes in AMPA/NMDA ratios throughout the fear circuit.</a> , (2018).</li><li>Eagle, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+prolonged+stress+enhances+hippocampal+glucocorticoid+receptor+and+phosphorylated+protein+kinase+B+levels.">Single prolonged stress enhances hippocampal glucocorticoid receptor and phosphorylated protein kinase B levels.</a> Neuroscience Research. 75 (2), 130-137 (2013).</li></ol>

The authors have nothing to disclose.

The results presented in this article show that near-infrared immunocytochemistry in combination with high-resolution scanning can be used to obtain semi-quantitative measures of protein expression in brain tissue. It can also be used to label two proteins simultaneously in the same brain region. We have previously used near-infrared immunohistochemistry to measure immediate early gene expression in multiple brain regions9,10. Immediate early genes can be used as a measure of neural activity. We also subjected these semi-quantitative measures of protein expression to statistical analyses that allowed us to group brain regions with correlated levels of immediate early genes (IEGs). We used this as a measure of functional connectivity to examine how stress affects functional connectivity among nodes within the fear circuit during emotional learning and memory9,10. We showed how AMPA/NMDA ratios (a signature of learning and memory) can be measured using near-infrared immunocytochemistry with high resolution scanning13. A similar technique can be used to measure pan and phospho-proteins to determine molecular signaling. This is accomplished using western blot14. However, this requires dissecting brain regions out of thick brain slices and is not amenable to small brain regions. This issue can be circumvented using near-infrared immunocytochemistry with high-resolution scanning. Finally, all immunocytochemistry images are digitized, which allows for unlimited storage and convenient re-analysis of previous assays.
As with all methods there are drawbacks. There is no magnification in high-resolution scanners and the treatment of tissue does not readily allow for probing using fluorescence microscopic techniques. Even taking alternate slices from the brain may not work, since confocal microscopy may work best in perfused brain tissue, but near-infrared imaging with high-resolution scanning is typically done on flash frozen brains. Protein expression in specific neurons (e.g., interneuron vs. pyramidal neuron) or different cell types in the brain (e.g., neurons vs. glia) cannot be determined using near-infrared immunocytochemistry with high-resolution scanning. Performing validation assays are critical as this is still a relatively new method for examining protein expression in brain tissue.
When used appropriately, near-infrared immunocytochemistry with high-resolution scanning offers advantages. Autofluorescence in brain tissue is reduced in the near-infrared range, semi-quantitative measures of protein expression can be obtained, expression of two proteins in the same brain region can be obtained, and images of the assay can be stored indefinitely. When paired with the correct protein and/or statistical method this technique can be used to examine protein expression, neural activity, molecular signaling, and functional connectivity within the brain.

The study of neuroscience concerns an investigation of how cells in the brain mediate specific functions1. These can be cellular in nature such as how glia cells confer immunity in the central nervous system or can involve experiments that aim to explain how the activity of neurons in the dorsal hippocampus leads to spatial navigation. In a broad sense, cellular function is determined by the proteins that are expressed in a cell and the activity of these proteins2. As a result, measuring the expression and/or activity of proteins in brain cells are critical for the study of neuroscience.
A number of techniques are available to measure protein expression in the brain. These include in vivo methods such as positron emission topography for receptor densities3 and micro-dialysis for small peptides4. More commonly, ex vivo methods are used to examine protein function and expression. These include mass spectrometry techniques5, western blot and enzyme-linked immunosorbent assay (ELISA)6, and immunocytochemistry7. Immunocytochemistry is widely used in the field of neuroscience. This technique involves the use of a primary antibody to detect a protein (or antigen) of interest (e.g., c-Fos) and a conjugated secondary antibody to detect the protein-primary antibody complex (Figure 1). To enable detection of the protein-primary antibody-secondary antibody complex, secondary antibodies have oxidizing agents such as horseradish peroxidase (HRP) conjugated to them. This allows for the formation of precipitates in cells that can be detected using light microscopy7. Secondary antibodies can also have chemicals fluoresce conjugated to them (i.e., fluorophores). When stimulated these chemicals emit light, which can be used to detect protein-primary antibody-secondary antibody complexes7. Lastly, sometimes primary antibodies have reducing agents and fluorescence chemicals attached to them directly negating the need for secondary antibodies7 (Figure 1).
Interestingly, many immunocytochemistry methods allow for visualization of proteins in brain cells, but not the ability to quantify the amount of protein in a specific cell or brain region. Using light microscopy to detect precipitates from reduction reactions allows for visualization of neurons and glia, but this method cannot be used to quantify protein expression in cells or in a specific brain region. In theory, fluorescence microscopy can be used for this, because the light emitted from the fluorescent secondary antibody is a measure of the protein-primary antibody-secondary antibody complex. However, autofluorescence in brain tissue can make it difficult to use fluorescence microscopy to quantify protein expression in brain tissue8. As a result, light emitted from fluorescent images of brain tissue is rarely used to quantify protein expression in the brain.
Many of these issues can be addressed using near-infrared immunocytochemistry in conjunction with high-resolution scanning9,10. In this article, we describe how immunocytochemistry coupled with fluorophores in the near-infrared emission spectra can be combined with high resolution scanning (e.g., 10&#8211;21 &#181;m) to obtain sharp images that allow for semi quantification of protein in distinct brain regions.

<table><tbody><tr><td>Brain Extraction</td><td></td><td></td><td></td></tr><tr><td>Anesthesia Induction Chamber</td><td>Kent Scientific</td><td>VetFlo-0530SM</td><td></td></tr><tr><td>Kleine Guillotine</td><td>Harvard Apparatus</td><td>73-1920</td><td></td></tr><tr><td>Friedman Rongeur</td><td>Fine Science Tools</td><td>16000-14</td><td>used to remove back of skull</td></tr><tr><td>Delicate Dissecting Scissors</td><td>Fischer Scientific</td><td>08-951-5</td><td>used to cut upward along midline of skull</td></tr><tr><td>Micro Spatula</td><td>Fischer Scientific</td><td>21-401-5</td><td>used to scoop out brain</td></tr><tr><td>Glass Microscope Slides</td><td>Fischer Scientific</td><td>12-549-6</td><td></td></tr><tr><td>Immunohistochemical Reaction</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Triton X-100</td><td></td><td></td><td>Used as a mild detergent to permeabilize cells after fixing in Paraformaldehyde, also used as mild detergent in combination with host serum and secondary antibody&amp;nbsp;</td></tr><tr><td>Tween-20</td><td></td><td></td><td>Used as a small amount of detergent added to TBS&amp;nbsp; to procuce TBS-T after coverslipping slides with primary antibody</td></tr><tr><td>Licor Odyssey scanner</td><td>Licor Biotechnology Inc.</td><td></td><td></td></tr><tr><td>Image Studio</td><td>Licor Biotechnology Inc.</td><td></td><td></td></tr></tbody></table>

using near-infrared fluorescence and high-resolution scanning to measure protein expression in the rodent brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Prior to using high-resolution scanning for immunohistochemistry, one should verify that the protocol works. This can be accomplished using a validation assay where brain sections from the same animal are incubated with primary and secondary antibodies, secondary antibody alone, or neither primary nor secondary antibody. Results for such a validation assay are shown in Figure 2. In this reaction we were detecting the immediate early gene c-Jun in the dorsal hippocampus and amygdala. C-Jun expression was only observed when primary and secondary antibodies were applied to the brain tissue.
Figure 3&#160;shows dual protein detection in amygdala nuclei. In this experiment we assayed the GluR1 subunit of the &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and the NR2A subunit of the N-methyl-D-aspartate receptor (NMDA) receptor in the same brain tissue. This allowed us to examine the ratio of AMPA/NMDA receptors in sub-nuclei of the amygdala. This ratio is a neurobiological signature of learning and memory11,12. As can be seen in Figure 3, signal for GluR2 (780 nm light pseudo colored in green) and NR2A (680 nm light pseudo colored in red) can only be observed in the brain tissue that was exposed to primary and secondary antibodies.&#160;
Figure 4&#160;shows how mean and normalized measures of protein expression can be obtained from high-resolution scans in the ventral hippocampus. Using the image analysis software, a rectangle is placed in the region of interest (molecular layer of CA1) and mean intensity of light from the shape can be used as a measure of fluorophore expression (Figure 4A). In turn, this is a measure of protein expression (i.e., the antigen-primary antibody-secondary antibody complex). The shape can also be placed across a region that expresses high signal and low signal to obtain a normalized curve (Figure 4B). In this example, a rectangle was placed across the molecular layer of CA1, but covered the dendritic fields as well, which did not express significant amounts of c-Jun in this assay. The area under the curve can then be used as a measure of protein expression. If the setup in an experiment involves treatment groups (e.g., stress exposure) and a control, relative changes in protein expression in the brain can be obtained.&#160;
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59685/59685fig1.jpg" /> 
Figure 1: Illustration of the immunohistochemical reaction using primary (1st) and secondary (2nd) antibodies or just primary antibody. Filled black circles represent a label, which could be an oxidizing agent such as horseradish peroxidase or a fluorophore such as boron-dipyrromethene (BODIPY). Green squares represent antigen (on protein of interest) being detected in the immunohistochemical reaction.&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59685/59685fig2v2.jpg" /> 
Figure 2: Images of a validation assay for detection of c-Jun. In this assay, tissue from the same animal was treated with a rabbit primary antibody that recognizes c-Jun and goat antirabbit secondary antibody with attached fluorophore with emission at 780 nm (pseudo colored in green, left panel). Adjacent tissue was treated with either secondary antibody alone (middle panel) or no antibody (right panel). Scale bar = 1 mm.&#160;
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59685/59685fig3.jpg" /> 
Figure 3: Validation assay for double labeling immunohistochemical reaction in the amygdala. Triplicate brain sections from the same rat was either exposed to rabbit antibody that recognizes GluR1 and mouse antibody that recognizes NR2A (left panels), secondary antibody (middle panels), or no antibody (right panels). GlurR1 was visualized with goat anti rabbit 800CW secondary antibody (780 nm, pseudo colored in green) and NR2A was visualized using a goat antimouse 680RD secondary antibody (680 nm, pseudo colored in red). Scale bar = 1 mm. ot = optic tract; ic = internal capsule; ec = external capsule. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59685/59685fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59685/59685fig4.jpg" /> 
Figure 4: Obtaining semi quantitative measures of protein expression in brain tissue.&#160;(A and B) screenshots of scored images in the image analysis software that is used to analyze images from the scanner. Tissue was from the ventral hippocampus and treated to visualize c-Jun.&#160;&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59685/59685fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain at JoVE.com

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

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Universidad San Sebastian

Research

JoVE Journal

Neuroscience

5.5K Views.  University of Delaware. Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Video: Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Medicine

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca2+-detector, TN-XXL. Activation of the receptor leads to an increase in the FRET signal. CNiFERs have nM sensitivity and a temporal response of seconds because a CNiFER clone utilizes the native receptor for a particular neurotransmitter, e.g., D2R for dopamine. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred &#181;m, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER.

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo

We present a protocol to create cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) for the optical detection of volumetric neurotransmitter release.

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

Quantifying the Distribution of a Presynaptic Protein in the Mouse Brain Using Immunofluorescence

Source: Wallrafen, R., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58940">Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence.</a> J. Vis. Exp. (2019).
This video demonstrates the immunofluorescence staining of a mouse brain slice to quantify the distribution of a specific presynaptic protein. The process involves blocking nonspecific sites, applying primary antibodies for the target protein and a reference marker, followed by secondary antibody labeling and nuclear counterstaining. The reference marker ensures accurate quantification by providing a consistent baseline, and the distribution of the presynaptic protein is analyzed across brain regions using confocal microscopy.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunofluorescence

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 Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections

<a href='https://app-jove-com.bdigitaluss.remotexs.co/v/53976/combining-double-fluorescence-situ-hybridization-with-immunolabelling'>Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections. J. Vis. Exp. (2016).</a>This video demonstrates the use of double fluorescence in situ hybridization to visualize the expression of two RNA targets in mouse brain sections. The protocol involves sequential detection of RNA using probes labeled with fluorescein isothiocyanate and digoxigenin, followed by enzymatic signal amplification to enhance sensitivity. The resulting fluorescence enables co-localization analysis of the target RNAs within the tissue.

Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

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A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Quantifying the Distribution of a Presynaptic Protein in the Mouse Brain Using Immunofluorescence

Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections