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.
1 . Mouse eye enucleation, eye cup dissection, and embedding
<ol>
	<li>Euthanize mice with carbon dioxide (CO2) followed by either decapitation (postnatal mice P0 - P11) or cervical dislocation (adult &#62;P11 mice). For P0-P14 mice, eyes are covered by skin. Ensure to remove the skin prior to subsequent steps.</li>
	<li>Mark the temporal part of the eye (Figure 2A) by slightly burning the cornea using a tail cauterizer. Avoid burning a hole in the cornea by touching the cornea very lightly and for no more than a split second with the cauterizer.</li>
	<li>Immediately enucleate mouse eyes using curved Dumont #5/45 forceps. Incubate for 15 min in a 1.5 mL cryotube tube with 1 mL of 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) on ice.</li>
	<li>After 15 min in 4% PFA, transfer the fixed eye using the curved forceps Dumont #5/45 to a modified 35 mm dissection dish (see Table of Materials and Figure 1C) filled with PBS and place under dissecting microscope.</li>
	<li>Make a small incision at the burn mark, perpendicular to and just above the limbus (border of the cornea) using the micro knife.</li>
	<li>Insert the curved scissors into the incision and perform a circumferential cut of the cornea following the limbus (Figure 2B).</li>
	<li>Remove the cornea and extract the lens with a thin Dumont #5 forceps, (Figure 2B) and lift it delicately away from the posterior portion of the eye. The vitreous body, the clear gel that fills the space between the lens and the retina, will come out with the lens (Figure 2B) and the retina will be visible as a white surface covering the inside of the posterior eye cup. Ensure that there is no visible damage to the retina, such as holes or tears (Figure 2C).</li>
	<li>Wash the eye cups twice in 1 mL of PBS for 10 min at room temperature.</li>
	<li>Cryoprotect the eye cups by equilibrating them in a solution of 15% sucrose in PBS overnight at 4 &#176;C until the eye cup sinks</li>
	<li>Transfer the eye cups to 30% sucrose in PBS for 2-3 h until the eye cup sinks.</li>
	<li>Embed the eye cups in OCT in 10 x 10 mm cryomolds (Figure 3A). Using a dissecting microscope, orient the eye in the cryomold along its dorsal-ventral axis (Figure 2D, Figure 3A) by rotating the eye until the burn mark is on top, the optic nerve is on the left and the cut-out part of the mold faces the right (Figure 2D). Take care to avoid bubbles near the tissue. 
	NOTE: To manipulate the eye cup, use a titanium probe made of a titanium wire attached to a pipette tip.</li>
	<li>To snap-freeze retinal tissue, immerse the cryomold for at least 5 min in a metal beaker containing isopentane and place the beaker in liquid nitrogen or dry ice/100% ethanol bath (up to 1/3 of the height of the metal beaker). 
	NOTE: The cryomold should be immersed in isopentane to greater than half of its height but does not have to be completely submerged.</li>
	<li>Remove the frozen block and wrap it in aluminum foil.</li>
	<li>Store at -80 &#176;C.</li>
	<li>Mark the frozen block using a pen or sharpie to record the orientation of the block (Figure 3B).</li>
</ol>
2 . Sectioning using a cryostat
<ol>
	<li>After adjusting the cryostat temperature (between -20 and -25 &#176;C), allow the molds containing the embedded eye cups to equilibrate to the cryostat temperature for 1 h.</li>
	<li>Install the block with dorso-ventral orientation (Figure 3C) and cut 10 &#181;m thick serial sections. Carefully place the retinal sections on annotated and numbered microscope slides.</li>
	<li>Store slides in slide boxes at -20 &#176;C or -80 &#176;C until IHC procedures.</li>
</ol>
3 . Fluorescence immunostaining using the Slide Rack
NOTE: The Slide Rack system (e.g., Sequenza) holds the glass microscope slides with a cover plate, creating a ~100 &#181;L capillary gap between the slide and the plate.
<ol>
	<li>Thaw the cryo-sections at room temperature (RT) for 2 h to 4 h to allow them to dry and attach to microscope slides. 
	NOTE: It is also possible to dry them at 30-37 &#176;C for 30 min to 1 h.</li>
	<li>Place slides in Slide Rack and wash the retinal sections twice in 100-200 &#181;L PBS for 2 min.</li>
	<li>Permeabilize the retinal sections by incubating them in 0.25% Triton-X / 0.05% NaN3in PBS for 5 min at RT.</li>
	<li>Add 100 &#181;L of blocking solution to the slides.</li>
	<li>Add 100 &#181;L of primary antibody diluted in blocking solution to the slides.</li>
	<li>Incubate retinal sections overnight at 4 &#730;C. 
	NOTE: During this time, cover the Slide Rack to avoid desiccation of the sections.</li>
	<li>Wash the retinal sections 3 times, each for 15 min, using 200 &#181;L PBS. 
	NOTE: The addition of 0.001- 0.002% Triton-X100 to PBS (PBS-T) allow a more stringent wash but can disrupt some membranes and cellular structures.</li>
	<li>Incubate retinal sections in fluorescent-labeled secondary antibodies for 1 h at RT. From now on protect from light.</li>
	<li>Wash the retinal sections 3 times, each for 15 min.</li>
	<li>Incubate the retinal sections at RT for 5 min with a nuclear marker such as Hoechst 33342 or DAPI solution.</li>
	<li>Wash the retinal sections 1 time for 15 min.</li>
	<li>Place a coverslip on top of a paper towel on the lab bench.</li>
	<li>Add 2 drops of anti-fading mounting media to the center of the coverslip.</li>
	<li>Turn the slide over to have the retinal cryo-section facing down.</li>
	<li>Carefully mount the coverslip slowly, at a ~45&#730; angle, tip the slide onto the mounting media. 
	NOTE: Avoiding forming bubbles as you lower the slide in place.</li>
	<li>Apply even pressure, using a heavy book or catalog (see below), to the slide-coverslip combination to eliminate excess mounting media. 
	NOTE: To ensure consistency in sample preparation, always use the same object, (i.e., the same book or catalog) to apply even pressure to the coverslip during slide mounting.</li>
	<li>Keep flat and allow to harden overnight at RT in the dark. Store slides flat at 4 &#176;C indefinitely.</li>
	<li>Image via wide field or confocal fluorescence microscopy (Figure 4).</li>
</ol>

<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>

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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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Neuroscience

26.8K Views.  University of Pennsylvania School of Veterinary Medicine. 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.

Video: Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...

Mouse Retina Isolation and Methanol Treatment for Studying Retinal Ganglion Cells

Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in RGCs have a close relationship with numerous retinal degenerative diseases. Whole-mount retinal immunostaining is frequently used in experimental studies on RGCs to evaluate the developmental and pathological conditions of the retina. Under some circumstances, some valuable retina samples, such as those from transgenic mice, may need to be retained for a long period without affecting the morphology or number of RGCs. For credible and reproducible experimental results, using an effective preserving medium is essential. Here, we describe the effect of methanol as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage. In brief, during the isolation process, cold methanol (&#8722;20 &#176;C) is pipetted onto the surface of the retina to help fix the tissues and facilitate their permeability, and then the retinas can be stored in cold methanol (&#8722;20 &#176;C) before being immunostained. This protocol describes the retina isolation workflow and tissue sample storage protocol, which is useful and practical for the investigation of RGCs.

Author Spotlight: Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells  

Methanol can be used as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage, which is useful for the investigation of retinal ganglion cells.

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in ...

Immunohistochemistry of Drosophila Larva for Microtubule Visualization

Mouse Retinal Dissection for Split Retina Application

Visualizing Retinal Synaptic Structures in 3D with Volume Electron Microscopy

Preparation of Retinal Samples for Volume Electron Microscopy

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level precision. Within the retina, various types of neurons establish synaptic connections in the inner and outer plexiform layers. While conventional EM techniques have yielded valuable insights into retinal subcellular organelles, their limitation lies in providing 2D image data, which can hinder accurate measurements. For instance, quantifying the size of three distinct synaptic vesicle pools, crucial for synaptic transmission, is challenging in 2D. Volume EM offers a solution by providing large-scale, high-resolution 3D data. It is worth noting that sample preparation is a critical step in Volume EM, significantly impacting image clarity and contrast. In this context, we outline a sample preparation protocol for the 3D reconstruction of photoreceptor axon terminals in the retina. This protocol includes three key steps: retina dissection and fixation, sample embedding processes, and selection of the area of interest.

Author Spotlight: Retinal Neuroscience Studies with Volume Electron Microscopy

This protocol describes the detailed steps for preparing retinal samples for volume electron microscopy, focusing on the structural features of retinal photoreceptor terminals.

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level ...

Optimized Protocol for Retinal Organoid Sample Preparation for TEM

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

Author Spotlight: Analyzing the Synaptic Ultrastructure in Mature Retinal Organoids Using TEM

This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem ...

Synaptic Circuits Analysis and Protein Localization in the Mouse Retina

Investigating Retinal Circuits and Molecular Localization by Pre&#45;Embedding Immunoelectron Microscopy

The retina comprises numerous cells forming diverse neuronal circuits, which constitute the first stage of the visual pathway. Each circuit is characterized by unique features and distinct neurotransmitters, determining its role and functional significance. Given the intricate cell types within its structure, the complexity of neuronal circuits in the retina poses challenges for exploration. To better investigate retinal circuits and cross-talk, such as the link between cone and rod pathways, and precise molecular localization (neurotransmitters or neuropeptides), such as the presence of substance P-like immunoreactivity in the mouse retina, we employed a pre-embedding immunoelectron microscopy (immuno-EM) method to explore synaptic connections and organization. This approach enables us to pinpoint specific intercellular synaptic connections and precise molecular localization and could play a guiding role in exploring its function. This article describes the protocol, reagents used, and detailed steps, including (1) retina fixation preparation, (2) pre-embedding immunostaining, and (3) post-fixation and embedding.

Author Spotlight: Understanding the Ultrastructural Basis of Retinal Synaptic Connectivity and Neurotransmitter Localization in Mice 

This protocol outlines the detailed steps of pre-embedding immunoelectron microscopy, with a focus on exploring synaptic circuits and protein localization in the retina.