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/pubmed?term=((Labelled+antigens+and+antibodies%5BTitle%5D)+AND+%22Annual+Review+of+Microbiology%22%5BJournal%5D)">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/pubmed?term=((Fluorescent+antibodies+as+histochemical+tools%5BTitle%5D)+AND+%22Federation+Proceedings%22%5BJournal%5D)">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/pubmed?term=((Localization+of+antigen+in+tissue+cells%3B+improvements+in+a+method+for+the+detection+of+antigen+by+means+of+fluorescent+antibody%5BTitle%5D)+AND+%22Journal+of+Experimental+Medicine%22%5BJournal%5D)">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/pubmed?term=((Artefacts+in+histopathology%5BTitle%5D)+AND+%22Journal+of+Oral+Maxillofacial+Pathology%22%5BJournal%5D)">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/pubmed?term=((Frozen+sections+from+Bouin-fixed+material+in+histopathology%5BTitle%5D)+AND+%22Stain+Technology%22%5BJournal%5D)">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/pubmed?term=((Application+of+alternative+fixatives+to+formalin+in+diagnostic+pathology%5BTitle%5D)+AND+%22European+Journal+of+Histochemistry%22%5BJournal%5D)">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/pubmed?term=((Application+of+cryoultramicrotomy+to+immunocytochemistry%5BTitle%5D)+AND+%22Journal+of+Microscopy%22%5BJournal%5D)">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/pubmed?term=((Molecular+properties+of+rhodopsin+and+rod+function%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Reexamination+of+Dopaminergic+Amacrine+Cells+in+the+Rabbit+Retina%3A+Confocal+Analysis+with+Double-+and+Triple-labeling+Immunohistochemistry%5BTitle%5D)+AND+%22Experimental+Neurobiology%22%5BJournal%5D)">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/pubmed?term=((GABA+and+Glutamate+Uptake+and+Metabolism+in+Retinal+Glial+%28Muller%29+Cells%5BTitle%5D)+AND+%22Frontiers+in+Endocrinology+%28Lausanne%29%22%5BJournal%5D)">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/pubmed?term=((Ptf1a+is+essential+for+the+differentiation+of+GABAergic+and+glycinergic+amacrine+cells+and+horizontal+cells+in+the+mouse+retina%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((The+murine+cone+photoreceptor%3A+a+single+cone+type+expresses+both+S+and+M+opsins+with+retinal+spatial+patterning%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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 commencing with dissection, it is important to be comfortable, relaxed and set the room lighting for optimal vision during mouse eye microdissection. To maintain mouse eye cup shape while dissecting the eye, we suggest keeping eyes submerged in PBS in custom made wax-coated dissection dishes. We suggest coating a 35mm dissection dish with light pink colored dental wax (Figure 1C). Indeed, the light pink color of the dental wax provides adequate contrast to see readily eye structures under a dissection scope, the eye-sized impressions in the wax, made by pressing the base of microfuge tubes into the wax, will keep the eye cup in place while allowing the appropriate rotation of the tools around the eye cup during the dissection. In addition, it is important to remove as much vitreous fluid from the eye cup as possible while removing the lens, as residual vitreous fluid is prone to form tissue damaging ice crystals during the tissue block freezing step. We generally use a tissue paper and place it to the border of the dissected eye cup, to carefully remove all remaining vitreous fluid by capillary without disturbing the retina tissue. In the unfortunate case where a small amount of residual vitreous fluid is still present in the eye cup, sectioning should be done at -20 &#176;C to reduce transformation of the vitreous in ice and therefore preserve the sharpness of the blade and the quality of the sectioning.
Fixation and embedding methods also have a major impact on the quality of the sections and the IHC. Although formalin-fixed paraffin-embedded sections yield optimal structural integrity of the eye tissue, formalin contains methanol that can enhance background fluorescence. To avoid these artifacts, we recommend using fresh EM grade paraformaldehyde (PFA) to limit background and autofluorescence caused by methanol or oxidized PFA4,5,6. We recommend the mild fixation conditions described in our protocol fixing the eye with 4% paraformaldehyde for 15 min on ice. Since PFA rapidly penetrates the tissue, but cross-links proteins very slowly, those conditions are sufficient to preserve retinal structural integrity during the dissection and preserve the antigenicity of common retinal antigens without the need for antigen retrieval methods4. If the retina structure is not well preserved, the duration of PFA fixation can be increased, however it is important to be aware that over-fixating tissues will enhance non-specific background fluorescence and can lead to epitope masking, while under-fixating tissues may compromise structural integrity of the tissue and cause the loss of some antigens prone to proteolytic degradation. Since short time PFA incubation is a relatively mild fixation, we also recommend snap-freezing retinas by dipping the OCT-embedded eye, contained in a cryomold (10 mm x 10 mm x 5 mm), in an isopentane bath immersed in liquid nitrogen. Snap-freezing tissues will preserve antigens, while sucrose solution and OCT act as cryo-protectants. This method will reduce the distortion of the tissue due to the formation of ice crystals, which can cause the so-called Swiss Cheese type of tissue damage. The cryomold should not be dipped directly in liquid nitrogen or it will start to boil, forming a vapor barrier and that will slow the freezing process, resulting in a heterogeneous fixation and preservation. Moreover, tissue and OCT in contact with liquid nitrogen can crack, thereby affecting the integrity of the eye7. We, therefore, recommend dipping the cryomold (10 mm x 10 mm x 5 mm) in no more than 5 mm deep isopentane using a small stainless-steel beaker, itself dipped in a larger stainless-steel beaker (100 mm wide minimum) containing liquid nitrogen. To avoid the rapid evaporation of the liquid nitrogen during the preparation of several samples, we keep both baths in an insulated cooler. It should be noted that cryopreserving tissues in OCT is not a long-term preservation method, due to the formation of ice crystals in the tissue over time which can alter subcellular morphology and/or denature some antigens. Consequently, tissue blocks and frozen sections should be stored at -20 &#176;C for short-term storage and for -80 &#176;C long-term storage.
The mammalian retina is an asymmetric organ with a specific spatial distribution of the cells across the temporal-nasal and dorso-ventral axes. For example, M- and S-opsin cones are arranged in a specific dorso-ventral gradient 12. Therefore, it is essential to ensure proper orientation of the eye throughout tissue preparation and sectioning in order to perform comparative IHC. We use a cauterizer to place a reference mark on the eye prior to dissection (Figure 2A, D) so that we can orient the eye cup along its dorsal-ventral axis during the embedding phase, and therefore obtaining perfectly transverse sections. We place the burn mark on the dorso-temporal part of the eye, by touching the cornea for a split second with the cauterizer. This will allow to slightly burn the cornea while avoiding piercing a hole into it. During the eye cup dissection, we make a small incision, at the burn mark, perpendicular to border of the cornea, using the micro knife (Figure 2B). To facilitate the dorso-ventral orientation of the block and minimize orientation adjustments during sectioning, we suggest orienting the posterior eye cup in the cryomold, with the burn mark facing the bottom of the cryomold and the opening of the cornea perfectly parallel of the cryomold&#39;s right side (Figure 3A, B). The frozen block will be installed with the bottom of the block facing toward the blade in the cryostat, the eye cup perpendicular to the blade, in a dorso-ventral orientation (Figure 3c).
Reliable retina IHC starts with optimal, adapted, and reproducible dissections and tissue processing. The method outlined here optimally preserves retinal structure and integrity while minimizing autofluorescence and epitope masking that can compromise fluorescent IHC. Following this protocol will ensure that you have reproducible high-quality data.

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><tbody><tr><td>6qt. Stainless Steel Beaker (185 x 218mm)</td><td>Gilson&amp;nbsp;</td><td>#MA-48</td><td>For liquid nitrogen (Figure 1E)</td></tr><tr><td>Aluminum foil&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Cauterizer&amp;nbsp;</td><td>Bovie</td><td>#AA01</td><td>To mark eye orientation (Figure 1A)</td></tr><tr><td>Curved forceps, Dumont #5/45 tweezers, 45-degrees bent&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>#72703-D</td><td>For mouse eye enucleation and maintenance (Figure 1A)</td></tr><tr><td>Curved scissors, Vannas Spring Scissors - 2.5mm Cutting Edge&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;</td><td>#15002-08</td><td>To circumferentially cut the cornea (Figure 1B)</td></tr><tr><td>Dental wax-coated 35mm dissection dish</td><td></td><td></td><td>For eye cup dissection (Figure 1C)</td></tr><tr><td>Dissecting microscope&amp;nbsp;</td><td>Leica, Houston, USA&amp;nbsp;</td><td>MZ12.5 High-performance stereomicroscope</td><td></td></tr><tr><td>Micro Knives - Plastic Handle&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;</td><td>#10315-12</td><td>To make a small incision at the burn mark (Figure 1A)</td></tr><tr><td>Pink dental wax&amp;nbsp;</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>#72660</td><td>For coating dissection dish</td></tr><tr><td>Slide Rack and Coverplate</td><td>Ted Pella</td><td>#36107</td><td>For retinal IHC (Figure 1G)</td></tr><tr><td>Stainless Steel Beaker (89 x 114mm)</td><td>Gilson&amp;nbsp;</td><td>#MA-40</td><td>For isopentane bath (Figure 1E)</td></tr><tr><td>Styrofoam box</td><td></td><td></td><td>For insulated cooler</td></tr><tr><td>Thin forceps Dumont #5&amp;nbsp;&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;</td><td>#11254-20</td><td>To remove the cornea and extract the lens (Figure 1B)</td></tr><tr><td>Tissue-Tek cryomold&amp;nbsp; (10mm x 10mm x 5mm)</td><td>Electron Miscroscopy Sciences&amp;nbsp;</td><td>#62534-10</td><td>Mold for OCT embedding</td></tr><tr><td>&lt;strong&gt;Buffers and Reagents&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Blocking solution</td><td></td><td></td><td>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.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Gelvatol (anti-fading mounting media)</td><td></td><td></td><td>Under the fume hood, dissolve 0.337g DABCO (Sigma cat #D2522-25G) in 10mL Fluoromount G (Fisher cat #OB100-01).&amp;nbsp; Adjust to pH 8-8.5 with 12N HCl (~ 5 drops).&amp;nbsp; Store in amber drop bottle at 4&amp;deg;C&amp;nbsp; (8).&amp;nbsp;</td></tr><tr><td>Hoechst 33342 1000x Stock</td><td>Thermo Scientific</td><td>#62249</td><td>1 mg/ml in PBS (1000x). Aliquot and store in dark, at 4&amp;deg;C for up to 1 year or at -20&amp;deg;C for long term storage. Prepare fresh&amp;nbsp; at 1 ug/mL (1x)&amp;nbsp;</td></tr><tr><td>Isopentane, 99%&amp;nbsp;</td><td>GFS Chemicals</td><td>#2961</td><td></td></tr><tr><td>Liquid Nitrogen</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde (PFA) in PBS</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>#15710</td><td>Prepared fresh 4% PFA from 16% PFA stock. Store the remaining 16% PFA at 4&amp;deg;C in the dark.&amp;nbsp;</td></tr><tr><td>Permeabilization solution</td><td></td><td></td><td>PBS + 0.25% Triton-X (AMRESCO cat #0694-1L) + 0.05% NaN3.&amp;nbsp; Prepare fresh.</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Bioworld&amp;nbsp;</td><td>#41620015-20</td><td>0.02 g/L KCl, 0.02 g/L KH&lt;sub&gt;2&lt;/sub&gt;PO4, 0.8 g/L NaCl, 0.216 g/L Na&lt;sub&gt;2&lt;/sub&gt;HPO4 , pH 7.4.&amp;nbsp; Prepared from 10x stock&amp;nbsp;</td></tr><tr><td>Sucrose solutions</td><td>Fisher Scientific&amp;nbsp;&amp;nbsp;</td><td>#S-5-500</td><td>Dissolve the appropriate amount of sucrose in&amp;nbsp; 37&amp;deg;C PBS (takes ~15 min to dissolve). May be stored for a few days at 4&amp;deg;C.&amp;nbsp;</td></tr><tr><td>Tissue-Tek OCT-compound&amp;nbsp;</td><td>Sakura</td><td>&amp;nbsp;#4583</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>anti-calbindin</td><td>Sigma-Aldrich</td><td>C8666</td><td>To label horinzotal cells&amp;nbsp;</td></tr><tr><td>anti-GAD65</td><td>Chemicon</td><td>AB5082</td><td>To label GABAergic amacrine cells</td></tr><tr><td>anti-GS</td><td>BD Transduction</td><td>610517</td><td>To label M&amp;uuml;ller cells</td></tr><tr><td>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). IHC indicate that our protocols consistently yield high quality and well-preserved retinal sections. Notably, the rhodopsin-rich inner and outer segments remain vertical and intact with little to no separation from overlying the retinal pigmented epithelium (RPE) (Figure 4A). In addition, M&#252;ller cells remain well-preserved with soma properly aligned and cytoplasmic processes that span from the ganglion cell layer (GCL) to the outer nuclear layer (ONL) (Figure 4B).
Interneurons, such as Gad65-positive amacrine cells, also remain properly stratified within the INL and inner plexiform layer (IPL) (Figure 4B) while well-defined horizontal cells are detectable at the INL and outer plexiform layer (OPL) border (Figure 4C). Collectively, these data demonstrate that our method preserves retinal cell and tissue integrity, from photoreceptors to ganglion cells
To facilitate transverse sectioning, it is important to meticulously orientate the optical cup in the cryo-mold prior to freezing. Misoriented retinas, poor sectioning technique or sectioning with dull or damaged microtome knives can cause artifacts such as retinal tissue tears, distortions, and cellular displacement, as in Figure 4. Examples of section artifacts are shown in Figure 4D-F. In Figure&#160;4D poor sectioning and alignment lead to photoreceptor inner and outer segments distortion (Figure 4D) and small tissue tears (Figure 4E, arrow). In another example, poor tissue orientation prior to section leads to sub-optimal alignment of Muller cell soma and cytoplasmic processes Figure 4E. Figure 4F shows how poor sectioning likely caused by a dull microtome knife caused horizontal cells to aberrantly smear from the OPL to the GCL. Thus, precise attention must be applied to the execution of each step outlined in the protocol to ensure the highest quality and reproducibility of IHC data.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59683/59683fig1.jpg" /> 
Figure 1: Tools for mouse eye dissection, embedding, and IHC. (A) Cauterizer; (B) Dissection tools from left to right: Dumont #5/45 curved forceps, Curved scissors, and Dumont #5 thin forceps; (C) 35 mm dissection dish (arrow points to dissecting chamber); (D) Dissecting microscope; (E) Frozen tools from left to right: Insulated cooler, stainless steel beaker for isopentane bath , stainless steel liquid N2 bath; (F) titanium probe; (G) Slide Rack and Coverplate for IHC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59683/59683fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59683/59683fig2.jpg" /> 
Figure 2: Mouse eye cup orientation and description. (A) Temporal burn mark location (red arrow) to facilitate eye orientation (dorso = D, ventral = V, temporal = T, nasal = N). (B) Schematic for mouse eye dissection. A small incision is made at the burn mark, perpendicular into the cornea and just above the limbus using a fine scalpel (Micro knife). Cut around the cornea to separate the anterior and posterior part of the eye. (C) Eye cup with an intact retina. (D) Dorso-ventral orientation of mouse eye with the optic nerve (ON), lens (L), cornea (C) the limbus (OS). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59683/59683fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59683/59683fig3.jpg" /> 
Figure 3: Mouse eye embedding. (A) Dorso-ventral orientated mice eye cup in cryomold filled with OCT, (B) Frozen mouse retinal cup in annotated cryomold. (C) Cryostat. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59683/59683fig3large.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/59683/59683fig4.jpg" /> 
Figure 4: Mouse retina immunohistochemistry.(A-C) Good quality and (D-F) poor quality retinal sections from P28 WT were labeled with antibodies to the rhodopsin (A, C) amacrine marker GAD65 (B, E) and the Mu&#776;ller cell marker glutamine synthetase (GS; B, E), and horizontal cell marker calbindin (C, F). White arrows point to tissue tears and aberrant smearing of the tissue. Scale bar, 20 &#181;m. Nuclei labeled with Hoechst 33342 (blue). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59683/59683fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

preparation-of-mouse-retinal-cryo-sections-for-immunohistochemistry

Nanyang Technological University

Research

JoVE Journal

Neuroscience

27.5K Views.  University of Pennsylvania School of Veterinary Medicine. Muis retinale dissectie is een echt delicaat proces als gevolg van de grootte en vorm van de ogen en de kwetsbaarheid van het netvlies sectie. Noodzakelijk om te oefenen, met een gedetailleerd protocol dat efficiënte methode en tips bieden is de sleutel tot hoge kwaliteit retinale dissectie en sectie. We geven tips en advies om onderzoekers te helpen veelvoorkomende valkuilen te vermijden en een aantal hoogwaardige netvliessecties te verkrijgen die geschikt zijn voor immunohistochemie.

Video: Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

Genetics

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining 

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

Biochemistry

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Biology

Mouse Dorsal Root Ganglion Cryosectioning

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of inflammatory and neuropathic pain, itch, as well as other peripheral neurological conditions. However, it remains a challenge to consistently obtain high-quality, intact, and flat cryostat sections onto glass slides because of the tiny sample size of the DRG tissue. So far, there is no article describing an optimal protocol for DRG cryosectioning. This protocol presents a step-by-step method to resolve the frequently encountered difficulties associated with DRG cryosectioning. The presented article explains how to remove the surrounding liquid from the DRG tissue samples, place the DRG sections on the slide facing the same orientation, and flatten the sections on the glass slide without curving up. Although this protocol has been developed for cryosectioning the DRG samples, it can be applied for the cryosectioning of many other tissues with a small sample size.

Author Spotlight: A Procedure for Mouse Dorsal Root Ganglion Cryosectioning  

Presented here is the development for consistently acquiring high-quality dorsal root ganglion cryostat sections.

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of ...

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 ...

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.

The retina comprises numerous cells forming diverse neuronal circuits, which constitute the first stage of the visual pathway. Each circuit is ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

Cryosectioning and Immunostaining of Mouse Retina

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. These methods enable detailed examinations of tissue morphologies and the localization of specific proteins within the tissue, which provide valuable insights into disease processes and mechanisms. Mice are the most widely used model for this purpose. However, because mouse eyeballs are small and mouse retinas are extremely delicate tissues, obtaining high-quality retinal sections and immunostaining images from mouse eyeballs is typically challenging. This study describes an improved protocol for cryosectioning mouse retinas and performing immunohistochemistry. An essential point of this protocol involves coating the eyeball with a layer of super glue, which prevents deformation of the eyeballs during the processes of cornea removal, lens extraction, and embedding. This step ensures the integrity of retinal morphologies is well preserved. This protocol highlights critical technical considerations and optimization strategies for consistently producing high-quality retinal sections and achieving excellent immunostaining results.

A protocol for preparing mouse retinal cryosections and performing immunostaining on photoreceptors is described. This article enables researchers to consistently produce mouse retinal frozen sections with well-preserved morphology and high-quality immunostaining results.

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

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