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 ...

Mouse retinal dissection is a really delicate process due to the size and shape of the eyes and the fragility of the retina section. Imperative to practice, having a detailed protocol that provide efficient method and tips is the key to high quality retinal dissection and section. We provide tips and advice to help researcher avoid common pitfall and obtain some high quality retinal sections suitable for immunohistochemistry.Mark the temporal part of the eye of a euthanized mouse using a tail cauterizer by touching the cornea very lightly and for no more than a split second to slightly burn the cornea and avoid making a hole. Immediately after that used curved Dumont 545 forceps to enucleate mouse eyes. Place the eyes in a 1.5 milliliter cryotube with one milliliter of 4%PFA and PBS and incubate for 15 minutes on ice.Then, transfer the fixed eyes using the curved Dumont 545 forceps to a modified 35 millimeter dissection dish filled with PBS and place under dissecting microscope. Use a microknife to make a small incision at the burn mark perpendicular to and just above the limbus border of the cornea. Insert curved scissors into the incision and perform a circumferential cut of the cornea following the limbus.Then, using thin Dumont 5 forceps, remove the cornea, extract the lens, and lift it delicately away from the posterior portion of the eye. The vitreous body will come out with the lens and the retina will be visible as a white surface covering the inside of the posterior eye cup. Wash the isolated eye cups twice in one milliliter of PBS for 10 minutes at room temperature.To cryo-protect the eye cups, equilibrate them in 15%sucrose and PBS overnight at four degrees Celsius until they sink. Then transfer the eye cups to 30%sucrose and PBS for two to three hours until they sink. Place the eye cups in OCT in 10 by 10 millimeter cryo-molds for embedding.Under a dissecting microscope, orient the eye in the cryo-mold along its dorsal ventral axis by rotating the eye until the burn mark is on top. The optic nerve is one the left and the cut out part of the mold faces the right. To snap freeze the retinal tissue, immerse the cryo-mold for at least five minutes in a metal beaker containing isopentane and then place the beaker in liquid nitrogen up to one third of the height of the beaker.Remove the frozen block, wrap it in aluminum foil, and store at minus 80 degrees Celsius. Use a pen or Sharpie to mark the frozen block to record its orientation. After adjusting the cryostat temperature between minus 20 and minus 25 degrees Celsius, place the molds containing the embedded eye cups inside and allow them to equilibrate to the cryostat temperature for one hour.Install the block in the cryostat with the dorsal ventral orientation and start cutting 10 micrometer thick serial sections. Carefully place the retinal sections on annotated and numbered microscope slides and then store in slide boxes at minus 20 degrees Celsius or minus 80 degrees Celsius until ready for immunostaining. Immunohistochemistry with antibodies to rhodopsin, a photoreceptor marker, glutamic acid decarboxylase 65, and amacrine cell marker, glutamine synthetase and Muller cell marker, and calbindin, a horizontal cell marker was performed on retinal sections.The results indicate that this protocol yields high quality and well preserved retinal sections unlike in poor quality retinal sections obtained from a different protocol. Rhodopsin rich inner and outer segments remain vertical and intact with little to no separation from overlying the retinal pigmented epithelium. Interneurons, such as Gad65 positive amacrine cells, remain properly stratified within the inner nuclear layer and inner plexiform layer.Also, Muller cells remain well preserved with soma properly aligned in cytoplasmic processes that span from the ganglion cell layer to the outer nuclear layer. Furthermore, well defined horizontal cells are detectable at the inner nuclear layer and outer plexiform layer border. It is important to mark the topper region of the eyes and keep the orientation during embedding.Always prepare paraformaldehyde under the hood with the appropriate personal protective equipment.

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

Research

JoVE Journal

Neuroscience

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 ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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 ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

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 ...

Detection of Abnormal Prion Protein by Immunohistochemistry

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform encephalopathies (TSEs). These neurodegenerative diseases affect humans and several animal species and include scrapie, zoonotic bovine spongiform encephalopathy (BSE), chronic wasting disease of cervids (CWD), and the newly identified camel prion disease (CPD). Diagnosis of TSEs relies on immunodetection of PrPSc by application of both immunohistochemistry (IHC) and western immunoblot methods (WB) on encephalon tissues, namely, the brainstem (obex level). IHC is a widely used method that uses&#160;primary antibodies (monoclonal or polyclonal) against antigens of interest in cells of a tissue section. The antibody-antigen binding can be visualized by a color reaction that remains localized in the area of the tissue or cell where the antibody was targeted. As such, in prion diseases, as in other fields of research, the immunohistochemistry techniques are not solely used for diagnostic purposes but also in pathogenesis studies. Such studies involve detecting the PrPSc patterns and types from those previously described to identify the new prion strains. As BSE can infect humans, it is recommended that biosafety laboratory level-3 (BSL-3) facilities and/or practices are used to handle cattle, small ruminants, and cervid samples included in the TSE surveillance. Additionally, containment and prion-dedicated equipment are recommended, whenever possible, to limit contamination. The PrPSc IHC procedure consists of a formic acid epitope-demasking step also acting as a prion inactivation measure, as formalin-fixed and paraffin-embedded tissues used in this technique remain infectious. When interpreting the results, care must be taken to distinguish non-specific immunolabeling from target labeling. For this purpose, it is important to recognize artifacts of immunolabeling obtained in known TSE-negative control animals to differentiate those from specific PrPSc immunolabeling types, which can vary between TSE strains, host species, and prnp genotype, further described herein.

Immunolabeling abnormal prion protein using immunohistochemistry protocols requires specific sample and anti-PrP antibody preparation methodologies. The present protocol describes the key steps in epitope demasking to ensure proper PrP immunolabeling and to minimize non-specific background staining. Also, this approach considers biosafety measures when conducting immunohistochemistry studies with the prion-infected tissues.

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform ...

Immunohistochemistry of Drosophila Larva for Microtubule Visualization

Remove the PBST and immerse the paraformaldehyde-fixed drosophila larval carcasses in a blocking agent for 40 minutes at room temperature. Replace the blocking agent with 200 microliters of the primary antibody diluted with 0.2%PBST. Leave the larvae to incubate at four degrees Celsius overnight.The next day, wash the larvae with 0.2%PBST for 10 minutes. Next, remove the PBST from the tube and add 200 microliters of diluted secondary antibody. Incubate it at ambient temperature for 1.5 hours in the dark.Introduce a nuclear dye such as TO-PRO R 3 iodide into the incubating tube for 30 minutes while staining the microtubules in the dark. Finally, rinse off the secondary antibody and the nuclear dye with 0.2%PBST for 10 minutes.