Name: preparation of mouse retinal cryo-sections for immunohistochemistry
Uploaded: 2019-07-01T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry at JoVE.com

This work was supported by funds from NIH (RO1-GMO97327) and the University Research Foundation (UPenn). We especially thank Svetlana Savina for her help in developing the immunohistochemistry protocol, Gordon Ruthel (University of Pennsylvania) and the Penn Vet Imaging Core for assistance with microscopy and Leslie King, Ph.D. for critical reading this manuscript.

All methods described here were carried out in strict accordance with the recommendations in the National Institutes of Health Guide for the Care and Use of Laboratory Animals provided by the Institute of Laboratory Animal Resources and were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.
All tools and equipment for the methods are shown in Figure 1 and listed in the Table of Materials.
<p class="...

<ol>
	<li>Coons, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labelled+antigens+and+antibodies.">Labelled antigens and antibodies.</a> Annual Review of Microbiology. 8, 333-352 (1954).</li><li>Coons, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+antibodies+as+histochemical+tools.">Fluorescent antibodies as histochemical tools.</a> Federation Proceedings. 10 (2), 558-559 (1951).</li><li>Coons, A. H., Kaplan, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+antigen+in+tissue+cells;+improvements+in+a+method+for+the+detection+of+antigen+by+means+of+fluorescent+antibody.">Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody.</a> Journal of Experimental Medicine. 91 (1), 1-13 (1950).</li><li>Chatterjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artefacts+in+histopathology.">Artefacts in histopathology.</a> Journal of Oral Maxillofacial Pathology. 18 (Suppl 1), S111-S116 (2014).</li><li>Hartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frozen+sections+from+Bouin-fixed+material+in+histopathology.">Frozen sections from Bouin-fixed material in histopathology.</a> Stain Technology. 20, 113 (1945).</li><li>Benerini Gatta, 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=Application+of+alternative+fixatives+to+formalin+in+diagnostic+pathology.">Application of alternative fixatives to formalin in diagnostic pathology.</a> European Journal of Histochemistry. 56 (2), e12 (2012).</li><li>Tokuyasu, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+cryoultramicrotomy+to+immunocytochemistry.">Application of cryoultramicrotomy to immunocytochemistry.</a> Journal of Microscopy. 143 (Pt 2), 139-149 (1986).</li><li>Imai, H., 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=Molecular+properties+of+rhodopsin+and+rod+function.">Molecular properties of rhodopsin and rod function.</a> Journal of Biological Chemistry. 282 (9), 6677-6684 (2007).</li><li>Lee, J. W., Lim, M. Y., Park, Y. S., Park, S. J., Kim, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reexamination+of+Dopaminergic+Amacrine+Cells+in+the+Rabbit+Retina:+Confocal+Analysis+with+Double-+and+Triple-labeling+Immunohistochemistry.">Reexamination of Dopaminergic Amacrine Cells in the Rabbit Retina: Confocal Analysis with Double- and Triple-labeling Immunohistochemistry.</a> Experimental Neurobiology. 26 (6), 329-338 (2017).</li><li>Bringmann, A., Grosche, A., Pannicke, T., Reichenbach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+and+Glutamate+Uptake+and+Metabolism+in+Retinal+Glial+(Muller)+Cells.">GABA and Glutamate Uptake and Metabolism in Retinal Glial (Muller) Cells.</a> Frontiers in Endocrinology (Lausanne). 4, 48 (2013).</li><li>Nakhai, H., 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=Ptf1a+is+essential+for+the+differentiation+of+GABAergic+and+glycinergic+amacrine+cells+and+horizontal+cells+in+the+mouse+retina.">Ptf1a is essential for the differentiation of GABAergic and glycinergic amacrine cells and horizontal cells in the mouse retina.</a> Development. 134 (6), 1151-1160 (2007).</li><li>Applebury, M. 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=The+murine+cone+photoreceptor:+a+single+cone+type+expresses+both+S+and+M+opsins+with+retinal+spatial+patterning.">The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning.</a> Neuron. 27 (3), 513-523 (2000).</li></ol>

Mouse retina dissection is a delicate process due to the small size and shape of mouse eyes and the fragility of retinal tissue. Even though performing high-quality dissection is a matter of practice, having a detailed protocol, providing efficient methods and tips is a necessity to obtain retinal sections and IHC. In addition to the protocols described here, there are several tips that allow for consistent high-quality retinal sections that are suitable for reproducible IHC.
Prior to commenci...

Immunohistochemistry (IHC) is a powerful technique for localizing specific proteins and cellular structures in tissues in situ1,2,3. Inappropriate fixation methods and sub-optimal sectioning of complex tissues can disrupt tissue structure, generate high background staining or diminish antibody-epitope interactions, resulting in staining artifacts and consequent misinterpretation of IHC data4. As the vertebrate retina is a complex and highly organized neural organ composed of strata of interconnected photoreceptors, interneurons and ganglion cells, it is very fragile and can be easily disrupted during dissection and sectioning. A detailed, standardized, and validated protocol from mouse eye dissection and orientation to immunostaining will help significantly reduce IHC artifacts, thereby, increasing the reliability of the results and allowing for more accurate comparative data analysis.
There are many protocols for tissue preparation for IHC, however, not all are suitable for retinal tissue. Strong fixatives like 10% formalin or Bouin&#39;s solution preserve retinal structure during dissection and sectioning5. Unfortunately, strong fixatives often lead to the enhanced background fluorescence and epitope masking due to the chemical modification of epitopes6. On the other hand, milder fixatives, like 4% paraformaldehyde (PFA) can alleviate some of these artifacts but require meticulous dissection and sectioning to preserve the optimal retinal structure. PFA rapidly penetrates tissue, but cross-links proteins very slowly, reducing the risk of epitope masking. Since short time PFA incubation is a relatively mild fixation, tissues often require rapid freezing to preserve antigens. It is important to avoid ice crystal formation during tissue freezing as they distort and damage the integrity of cells and tissues7.
Here we describe detailed and standardized protocols for dissection, fixation, and cryo-protection of mouse ocular posterior cups that yield consistent and reliable IHC data.

<table border="0" cellpadding="0" cellspacing="0" style="width:1128px;" width="1129">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">6qt. Stainless Steel Beaker (185 x 218mm)</td>
			<td style="width:209px;">Gilson&#160;</td>
			<td style="width:236px;">#MA-48</td>
			<td style="width:365px;">For liquid nitrogen (Figure 1E)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Aluminum foil&#160;</td>
			<td style="width:209px;"></td>
			<td style="width:236px;"></td>
			<td style="width:365px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Cauterizer&#160;</td>
			<td style="width:209px;">Bovie</td>
			<td style="width:236px;">#AA01</td>
			<td style="width:365px;">To mark eye orientation (Figure 1A)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Curved forceps, Dumont #5/45 tweezers, 45-degrees bent&#160;</td>
			<td style="width:209px;">Electron Microscopy Sciences</td>
			<td style="width:236px;">#72703-D</td>
			<td style="width:365px;">For mouse eye enucleation and maintenance (Figure 1A)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Curved scissors, Vannas Spring Scissors - 2.5mm Cutting Edge&#160;</td>
			<td style="width:209px;">Fine Science Tools&#160;</td>
			<td style="width:236px;">#15002-08</td>
			<td style="width:365px;">To circumferentially cut the cornea (Figure 1B)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Dental wax-coated 35mm dissection dish</td>
			<td style="width:209px;"></td>
			<td style="width:236px;"></td>
			<td style="width:365px;">For eye cup dissection (Figure 1C)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Dissecting microscope&#160;</td>
			<td style="width:209px;">Leica, Houston, USA&#160;</td>
			<td style="width:236px;">MZ12.5 High-performance stereomicroscope</td>
			<td style="width:365px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Micro Knives - Plastic Handle&#160;</td>
			<td style="width:209px;">Fine Science Tools&#160;</td>
			<td style="width:236px;">#10315-12</td>
			<td style="width:365px;">To make a small incision at the burn mark (Figure 1A)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Pink dental wax&#160;</td>
			<td style="width:209px;">Electron Microscopy Sciences&#160;</td>
			<td style="width:236px;">#72660</td>
			<td style="width:365px;">For coating dissection dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Slide Rack and Coverplate</td>
			<td style="width:209px;">Ted Pella</td>
			<td style="width:236px;">#36107</td>
			<td style="width:365px;">For retinal IHC (Figure 1G)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Stainless Steel Beaker (89 x 114mm)</td>
			<td style="width:209px;">Gilson&#160;</td>
			<td style="width:236px;">#MA-40</td>
			<td style="width:365px;">For isopentane bath (Figure 1E)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Styrofoam box</td>
			<td style="width:209px;"></td>
			<td style="width:236px;"></td>
			<td style="width:365px;">For insulated cooler</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Thin forceps Dumont #5&#160;&#160;</td>
			<td style="width:209px;">Fine Science Tools&#160;</td>
			<td style="width:236px;">#11254-20</td>
			<td style="width:365px;">To remove the cornea and extract the lens (Figure 1B)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">Tissue-Tek cryomold&#160; (10mm x 10mm x 5mm)</td>
			<td style="width:209px;">Electron Miscroscopy Sciences&#160;</td>
			<td style="width:236px;">#62534-10</td>
			<td style="width:365px;">Mold for OCT embedding</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Buffers and Reagents</td>
			<td style="width:209px;">Company</td>
			<td style="width:236px;">Catalog Number</td>
			<td style="width:365px;">Comments</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:317px;">Blocking solution</td>
			<td style="width:209px;"></td>
			<td style="width:236px;"></td>
			<td style="width:365px;">2% Normal Horse Serum, 1.5% Cold Fish skin Gelatin (at 40-50% in H2O, cat #G7765), 5% bovine serum albumin (cat #AK1391-0100) in permeabilization buffer.&#160;&#160;</td>
		</tr>
		<tr height="77">
			<td height="77" style="height:77px;width:317px;">Gelvatol (anti-fading mounting media)</td>
			<td style="width:209px;"></td>
			<td style="width:236px;"></td>
			<td style="width:365px;">Under the fume hood, dissolve 0.337g DABCO (Sigma cat #D2522-25G) in 10mL Fluoromount G (Fisher cat #OB100-01).&#160; Adjust to pH 8-8.5 with 12N HCl (~ 5 drops).&#160; Store in amber drop bottle at 4&#176;C&#160; (8).&#160;</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:317px;">Hoechst 33342 1000x Stock</td>
			<td style="width:209px;">Thermo Scientific</td>
			<td style="width:236px;">#62249</td>
			<td style="width:365px;">1 mg/ml in PBS (1000x). Aliquot and store in dark, at 4&#176;C for up to 1 year or at -20&#176;C for long term storage. Prepare fresh&#160; at 1 ug/mL (1x)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Isopentane, 99%&#160;</td>
			<td style="width:209px;">GFS Chemicals</td>
			<td style="width:236px;">#2961</td>
			<td style="width:365px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Liquid Nitrogen</td>
			<td style="width:209px;"></td>
			<td style="width:236px;"></td>
			<td style="width:365px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:317px;">Paraformaldehyde (PFA) in PBS</td>
			<td style="width:209px;">Electron Microscopy Sciences&#160;</td>
			<td style="width:236px;">#15710</td>
			<td style="width:365px;">Prepared fresh 4% PFA from 16% PFA stock. Store the remaining 16% PFA at 4&#176;C in the dark.&#160;</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:317px;">Permeabilization solution</td>
			<td style="width:209px;"></td>
			<td style="width:236px;"></td>
			<td style="width:365px;">PBS + 0.25% Triton-X (AMRESCO cat #0694-1L) + 0.05% NaN3.&#160; Prepare fresh.</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:317px;">Phosphate-buffered saline (PBS)</td>
			<td style="width:209px;">Bioworld&#160;</td>
			<td style="width:236px;">#41620015-20</td>
			<td style="width:365px;">0.02 g/L KCl, 0.02 g/L KH2PO4, 0.8 g/L NaCl, 0.216 g/L Na2HPO4 , pH 7.4.&#160; Prepared from 10x stock&#160;</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:317px;">Sucrose solutions</td>
			<td style="width:209px;">Fisher Scientific&#160;&#160;</td>
			<td style="width:236px;">#S-5-500</td>
			<td style="width:365px;">Dissolve the appropriate amount of sucrose in&#160; 37&#176;C PBS (takes ~15 min to dissolve). May be stored for a few days at 4&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Tissue-Tek OCT-compound&#160;</td>
			<td style="width:209px;">Sakura</td>
			<td style="width:236px;">&#160;#4583</td>
			<td style="width:365px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Antibodies</td>
			<td style="width:209px;">Company</td>
			<td style="width:236px;">Catalog Number</td>
			<td style="width:365px;">Comments</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:317px;">anti-calbindin</td>
			<td>Sigma-Aldrich</td>
			<td>C8666</td>
			<td>To label horinzotal cells&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:317px;">anti-GAD65</td>
			<td>Chemicon</td>
			<td>AB5082</td>
			<td>To label GABAergic amacrine cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:317px;">anti-GS</td>
			<td>BD Transduction</td>
			<td>610517</td>
			<td>To label M&#252;ller cells</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:317px;">anti-rhodopsin</td>
			<td>Millipore</td>
			<td>MAB5316</td>
			<td>To label rods</td>
		</tr>
	</tbody>
</table>

preparation of mouse retinal cryo-sections for immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

To illustrate how these protocols, ensure optimal retinal preservation for IHC, we probed retinal sections from P28 WT mice (C57BL/6N) with antibodies to rhodopsin (a photoreceptor marker)8, glutamic acid decarboxylase 65 (Gad65, an amacrine cell marker)9, glutamine synthetase (GS, a M&#252;ller cell marker)10, and calbindin (a horizontal cell marker)11&#160;(Figure 4</stro...

Watch this Scientific Journal Video about Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry at JoVE.com

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

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