This work was funded by the Hubei Key Laboratories Opening Project (grant no. 2021KFY055), Natural Science Foundation of Hubei Province (grant no. 2020CFB240), and Fundamental Research Funds for the Central Universities (grant no. 2042020kf0065).

All steps are performed at room temperature unless otherwise indicated. All C57BL/6J mice used were obtained from the Laboratory Animal Center of Wuhan University, and all the related experiments were approved by the Committee on the Ethics of Animal Experiments of Wuhan University. All efforts were made to minimize the suffering of the mice.
1. Enucleation and fixation of the eyes
<ol>
	<li>Euthanize the mice with carbon dioxide asphyxiation, and enucleate the eyeball gently using toothless tweezers.</li>
	<li>Rinse the eyeball once in phosphate-buffered saline (PBS).</li>
	<li>Puncture the eyeball with a syringe needle at the cornea under a microscope (eyepiece WF: 10x/22; continuous magnification objective: 0.67x-4.5x) to accelerate the fixation into the eyeball and improve the fixing efficiency.</li>
	<li>Transfer to a 24-well plate containing 4% paraformaldehyde (PFA).</li>
	<li>Fix the eyeball in 4% PFA for 45 min, and then transfer it to 1x PBS to remove excess PFA (wash three times, 5 min/time) immediately.</li>
</ol>
2. Retina isolation and flattening
<ol>
	<li>Transfer the eyeball to a glass slide, and place it under a dissecting microscope (eyepiece WF: 10x/22; continuous magnification objective: 0.67x-4.5x).</li>
	<li>Hold the forceps in one hand, and use it to clamp the margin of the cornea to prevent eyeball movement. Use the other hand to hold the dissecting scissors and cut around the cornea at approximately 1 mm of depth through the corneoscleral limbus. Remove the lens and vitreous body.</li>
	<li>Hold the optic disc at the center, and using dissecting scissors, cut the retina radially along the four quadrants, reaching approximately 2/3 of the radius of the retina.</li>
	<li>Clamp one of the scleral edges using toothed forceps. With the other hand, hold the edge of the sclera with untoothed forceps, and move from where the sclera is fixed by the toothed forceps toward the base of the sclera. Carefully peel the retina.</li>
	<li>Draw off PBS using a pipette (200 &#181;L) to flush and wet the retina, and then remove any excess PBS with a small piece of absorbent paper. If needed, gently flatten the retina with a small bristle brush.</li>
	<li>Slowly pipette cold methanol (precooled in a &#8722;20&#176;C freezer) drop by drop onto the inner surface of the retina (the volume of methanol is not fixed, as long as the retina can be completely coveredby the methanol). The retina turns white and develops increased tenacity.</li>
	<li>Gently flatten the retina, and remove impurities such as residual pigment membranes and the vitreous body with small bristle brushes. Invert the retina using small bristle brushes, and repeat the above-mentioned process.</li>
	<li>Transfer the methanol-treated retina into a well of a 24-well plate, and keep it soaked in methanol.</li>
	<li>Store it at &#8722;20&#176;C for 1-2 h for immediate immunostaining, or leave the retina in methanol, and store it at &#8722;20&#176;C for long-term storage.</li>
	<li>Remove the methanol from the 24-well plate, and rinse the retina with 1x PBS.</li>
</ol>
3. Immunofluorescent staining of the RGCs
<ol>
	<li>Carefully transfer the retina to a 24-well hemagglutination plate with a plastic Pasteur pipette, and block in 5% PBS-TX-BSA (5 g of bovine serum albumin powder dissolved in 100 mL of 1x PBST solution; the PBST is formed by adding 10 mL of Triton X-100 to 2,000 mL of 1 x PBS) at room temperature for 4 h or overnight at 4 &#176;C. Keep the ganglion side up.</li>
	<li>Remove the PBS-TX-BSA, and incubate the retina at 4 &#176;C for 48-96 h, adding 100 &#181;L of the selected primary antibody (guinea pig anti-RBPMS antibody) using a 1:400 dilution.</li>
	<li>Remove the primary antibody, and rinse the retina three times (20 min each) with 1x PBS, shaking at room temperature.</li>
	<li>Incubate the retina with gentle shaking overnight at 4 &#176;C with 100 &#181;L of the corresponding secondary antibody (see Table of Materials), cyanine Cy3 affiniPure donkey anti-guinea pig IgG (H+L), using a 1:400 dilution.</li>
	<li>Rinse the retina with 1x PBS three times (20 min each).</li>
	<li>Lay the retina flat on the slide with the ganglion cell layer facing upward, drop an appropriate amount of anti-fluorescence quenching agent around the retina with a pipette, and cover the slide with a coverslip (be careful not to have bubbles). 
	NOTE: To determine the appropriate amount of anti fluorescence quencher, ensure that it covers the entire retina. After that, cover the slide without allowing the retina to float and move easily.</li>
	<li>Seal the slide all around with a sealing agent to prevent it from moving. Store the samples at &#8722;20&#176;C, and protect them from light.</li>
	<li>Image the retina using a fluorescence/confocal microscope.</li>
</ol>

Mouse Retina Isolation and Methanol Treatment for Studying Retinal Ganglion Cells

<ol>
	<li>Sanes, J. R., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+types+of+retinal+ganglion+cells:+Current+status+and+implications+for+neuronal+classification.">The types of retinal ganglion cells: Current status and implications for neuronal classification.</a> Annual Review of Neuroscience. 38, 221-246 (2015).</li><li>Almasieh, M., Wilson, A. M., Morquette, B., Cueva Vargas, J. L., Di Polo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+retinal+ganglion+cell+death+in+glaucoma.">The molecular basis of retinal ganglion cell death in glaucoma.</a> Progress in Retinal and Eye Research. 31 (2), 152-181 (2012).</li><li>Au, N. P. B., Ma, C. H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation,+microglia+and+implications+for+retinal+ganglion+cell+survival+and+axon+regeneration+in+traumatic+optic+neuropathy.">Neuroinflammation, microglia and implications for retinal ganglion cell survival and axon regeneration in traumatic optic neuropathy.</a> Frontiers in Immunology. 13, 860070 (2022).</li><li>Pavlidis, M., Stupp, T., Naskar, R., Cengiz, C., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+ganglion+cells+resistant+to+advanced+glaucoma:+A+postmortem+study+of+human+retinas+with+the+carbocyanine+dye+DiI.">Retinal ganglion cells resistant to advanced glaucoma: A postmortem study of human retinas with the carbocyanine dye DiI.</a> Investigative Ophthalmology &amp; Visual Science. 44 (12), 5196-5205 (2003).</li><li>Vidal-Sanz, M., 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=Understanding+glaucomatous+damage:+anatomical+and+functional+data+from+ocular+hypertensive+rodent+retinas.">Understanding glaucomatous damage: anatomical and functional data from ocular hypertensive rodent retinas.</a> Progress in Retinal and Eye Research. 31 (1), 1-27 (2012).</li><li>Kole, C., 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=Activating+transcription+factor+3+(ATF3)+protects+retinal+ganglion+cells+and+promotes+functional+preservation+after+optic+nerve+crush.">Activating transcription factor 3 (ATF3) protects retinal ganglion cells and promotes functional preservation after optic nerve crush.</a> Investigative Ophthalmology &amp; Visual Science. 61 (2), 31 (2020).</li><li>Nadal-Nicol&#225;s, F. M., 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=Brn3a+as+a+marker+of+retinal+ganglion+cells:+qualitative+and+quantitative+time+course+studies+in+naive+and+optic+nerve-injured+retinas.">Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas.</a> Investigative Ophthalmology &amp; Visual Science. 50 (8), 3860-3868 (2009).</li><li>Kwong, J. M., Caprioli, J., Piri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+binding+protein+with+multiple+splicing:+A+new+marker+for+retinal+ganglion+cells.">RNA binding protein with multiple splicing: A new marker for retinal ganglion cells.</a> Investigative Ophthalmology &amp; Visual Science. 51 (2), 1052-1058 (2010).</li><li>Stradleigh, T. W., Ishida, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+strategies+for+retinal+immunohistochemistry.">Fixation strategies for retinal immunohistochemistry.</a> Progress in Retinal and Eye Research. 48, 181-202 (2015).</li><li>Stradleigh, T. W., Greenberg, K. P., Partida, G. J., Pham, A., Ishida, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moniliform+deformation+of+retinal+ganglion+cells+by+formaldehyde-based+fixatives.">Moniliform deformation of retinal ganglion cells by formaldehyde-based fixatives.</a> Journal of Comparative Neurology. 523 (4), 545-564 (2015).</li><li>Bucher, D., Scholz, M., Stetter, M., Obermayer, K., Pfl&#252;ger, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+methods+for+three-dimensional+reconstructions+from+confocal+images:+I.+Tissue+shrinking+and+axial+scaling.">Correction methods for three-dimensional reconstructions from confocal images: I. Tissue shrinking and axial scaling.</a> Journal of Neuroscience Methods. 100 (1-2), 135-143 (2000).</li><li>Chidlow, G., Daymon, M., Wood, J. P., Casson, R. J. <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+a+wide-ranging+panel+of+antigens+in+the+rat+retina+by+immunohistochemistry:+Comparison+of+Davidson's+solution+and+formalin+as+fixatives.">Localization of a wide-ranging panel of antigens in the rat retina by immunohistochemistry: Comparison of Davidson's solution and formalin as fixatives.</a> Journal of Histochemistry &amp; Cytochemistry. 59 (10), 884-898 (2011).</li><li>Tokuda, K., 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=Optimization+of+fixative+solution+for+retinal+morphology:+A+comparison+with+Davidson's+fixative+and+other+fixation+solutions.">Optimization of fixative solution for retinal morphology: A comparison with Davidson's fixative and other fixation solutions.</a> Japanese Journal of Ophthalmology. 62 (4), 481-490 (2018).</li><li>Miki, M., Ohishi, N., Nakamura, E., Furumi, A., Mizuhashi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+fixation+of+the+whole+bodies+of+fish+by+a+double-fixation+method+with+formalin+solution+and+Bouin's+fluid+or+Davidson's+fluid.">Improved fixation of the whole bodies of fish by a double-fixation method with formalin solution and Bouin's fluid or Davidson's fluid.</a> Journal of Toxicologic Pathology. 31 (3), 201-206 (2018).</li><li>Zanini, C., Gerbaudo, E., Ercole, E., Vendramin, A., Forni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+two+commercial+and+three+home-made+fixatives+for+the+substitution+of+formalin:+A+formaldehyde-free+laboratory+is+possible.">Evaluation of two commercial and three home-made fixatives for the substitution of formalin: A formaldehyde-free laboratory is possible.</a> Environmental Health. 11, 59 (2012).</li><li>Tang, M., 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=An+optimized+method+to+visualize+the+goblet+cell-associated+antigen+passages+and+identify+goblet+cells+in+the+intestine,+conjunctiva,+and+airway.">An optimized method to visualize the goblet cell-associated antigen passages and identify goblet cells in the intestine, conjunctiva, and airway.</a> Immunobiology. 227 (6), 152260 (2022).</li><li>Brock, R., Hamelers, I. H., Jovin, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+fixation+protocols+for+adherent+cultured+cells+applied+to+a+GFP+fusion+protein+of+the+epidermal+growth+factor+receptor.">Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor.</a> Cytometry. 35 (4), 353-362 (1999).</li><li>Baykal, B., Korkmaz, C., Kocabiyik, N., Ceylan, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+post-fixation+on+visualising+vimentin+in+the+retina+using+immunofluorescence+method.">The influence of post-fixation on visualising vimentin in the retina using immunofluorescence method.</a> Folia Morphologica. 77 (2), 246-252 (2018).</li><li>Powner, M. B., 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=Visualization+of+gene+expression+in+whole+mouse+retina+by+in+situ+hybridization.">Visualization of gene expression in whole mouse retina by in situ hybridization.</a> Nature Protocols. 7 (6), 1086-1096 (2012).</li><li>Zhang, N., Cao, W., He, X., Xing, Y., Yang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+methanol+to+preserve+retinas+for+immunostaining.">Using methanol to preserve retinas for immunostaining.</a> Clinical and Experimental Ophthalmology. 50 (3), 325-333 (2022).</li><li>Kalesnykas, G., 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=Retinal+ganglion+cell+morphology+after+optic+nerve+crush+and+experimental+glaucoma.">Retinal ganglion cell morphology after optic nerve crush and experimental glaucoma.</a> Investigative Ophthalmology &amp; Visual Science. 53 (7), 3847-3857 (2012).</li><li>Parrilla-Reverter, G., 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=Time-course+of+the+retinal+nerve+fibre+layer+degeneration+after+complete+intra-orbital+optic+nerve+transection+or+crush:+a+comparative+study.">Time-course of the retinal nerve fibre layer degeneration after complete intra-orbital optic nerve transection or crush: a comparative study.</a> Vision Research. 49 (23), 2808-2825 (2009).</li></ol>

The authors are not aware of any affiliations, memberships, funding, or financial holdings that might affect the objectivity of this study.

Fixation is an essential step to preserve the retina, which can impact any subsequent RGC investigations based on morphology. Successful fixation rapidly captures the structure and state of the retinas at the moment of exposing them to the fixing medium, which is critical for further analysis. Although formaldehyde has been regarded as one of the most common fixing agents for tissue and cell fixation and preservation, formaldehyde alone does not always work well as the optimal chemical fixative for some investigations10,14. Different kinds of fixatives have varying capacities to precipitate amino acids and nucleotides, stabilize proteins, and maintain cell morphology15. By using mixtures of both types of fixative agents, the strengths of each can be fully utilized16,17. Methanol is frequently used in many fixation procedures before the tissue is freshly frozen or embedded in ideal cutting materials16,18. For whole-mount experiments, 100% cold (&#8722;20 &#176;C) methanol is used to achieve retinal sample fixation, which aids the sample permeabilization for the subsequent fluorescence-based imaging19.
A key advantage of methanol fixation is that it allows for easy preservation. In this protocol, methanol is added to the front and back of the retina progressively at &#8722;20 &#176;C after the retina has been flattened. It can be easily observed that the color of the retina rapidly changes from translucent to milky white, and the impurities become clearer. It should be noted that the retina should be gently flattened with a brush to prevent curling around it during the methanol-dripping process. After the methanol treatment, the overall toughness of the retina increases, reducing the difficulty of removing the impurities and transferring the retina. Post-fixation with methanol is more convenient for preserving retinas that have been slightly pre-fixed with PFA. However, when the tenacity of the retina is improved, the tenacity of the vitreous and other impurities is also likely to increase accordingly. One can try to gently remove some impurities that are easy to remove before using methanol, and then removing the remaining impurities after drip-adding methanol, which is more easier to perform. The early removal of impurities requires enough practice and experience. It is recommended to gently brush the remaining vitreous body on the retina radially outward from the optic disc.
In this protocol, after the treatment, the retina is soaked in methanol and placed in a &#8722;20&#176;C freezer for some time before the subsequent sealing, and other treatments could further increase the toughness of the retina. However, it is difficult to state a precise length of time that would be universally optimal for immersion fixation. If needed, the retina could be stored in cold methanol for long periods.
In this work, with the presented methodology, the impact of storage in methanol on RGC staining - a crucial technique in retinal qualitative and quantitative evaluations - was examined. with the use of methanol, the fluorescence intensity of the RGCs, which would affect the outcomes of qualitative and quantitative testing, were not altered during immunostaining. The retinas could be kept indefinitely in a preserving solution, cold (&#8722;20&#176;C) methanol, for at least 12 months before staining. In addition, our previous study revealed no significant difference in RGC survival rate in the optic nerve crush mouse model following long-term storage in methanol, and methanol preservation also had little impact on the quantitative analysis of the retinal vasculature and retinal hypoxia in the oxygen-induced retinopathy model20. We recommend using 4% PFA and cold (&#8722;20&#176;C) methanol sequentially for sample preparation, as well as adopting methanol fixation for storage strategies in the laboratory. One limitation is that the analyses do not fully demonstrate subtle morphological changes at the dendrite or axonal level, though the density of the RGCs and the morphology of the soma do not seem to change21,22. It should be noted that, in this protocol, we only tested RBPMS for RGCs. Further verifying different RGC proteins (e.g., Brn3a, neuronal nuclei, neurofilament-L) would provide a more robust result. Moreover, the protocol also has the potential to be applied to any other cell type in the retina that needs further research. However, long-term inhalation or direct cutaneous exposure to methanol may cause toxic effects on the human body. Therefore, it is necessary to take good precautions in the process of using methanol.
Our method places a strong emphasis on using methanol fixation for the storage of retinas for RGC investigations and highlights important considerations regarding the RGC quantification, and fluorescence intensity. This protocol provides a straightforward and practical method to satisfy the necessity of preserving retinal samples for further use.

Retinal ganglion cells (RGCs) are the only projection neurons in the retina, and they integrate and transmit outside visual information to the brain1. Many neurodegenerative diseases such as glaucoma and traumatic optic neuropathy are characterized by irreversible damage and loss of RGCs2,3. Analyzing the morphological and quantitative changes of RGCs is a crucial step in determining how neurodegenerative diseases develop and advance4,5.
Indirect immunofluorescence assay is a widely accepted method to monitor the distribution of proteins and cell counting. In the laboratory, whole-mount retinal immunostaining is commonly used in experimental studies on RGCs to evaluate the physiological and pathological conditions of the retina6. The most common markers used for RGC quantification in the whole retina include Brn3a, RNA binding protein with multiple splicing (RBPMS), and so on7,8. Characterizing the amount and distribution of RGCs requires high-quality whole-mount retinal immunostaining. Generally, in immunostaining protocols, the retina is immersed in chemical fixatives before being incubated in antibodies. Ideal fixatives should not change the shape of cells, the accessibility or affinity of the epitopes for antibodies, or the linear dimensions of the tissue9,10. 
Due to the complex structure of the retina, problems such as retinal fragility and folding, as well as some common difficulties including cell shrinkage and unclear nuclei, are prone to occur when making a whole-mount retinal patch, which has a negative impact on experimental research. In addition, not all retinas are immunostained immediately, especially when it comes to the retinas of transgenic mice with expensive prices that are of precious origin, necessitating the preservation of the extra retinal samples for further use.
The appropriate fixative solution can fix the tissue quickly, avoid tissue autolysis, preserve the normal morphology and structure of the tissue cells, and keep the antigenicity of proteins and other substances10. At present, formaldehyde-based fixation has been widely used in various tissues, including separated retinas, hemisected eyecups, and whole eyeballs10. Tissue shrinkage and the morphological alteration of cells are the two critical challenges encountered following immersion in formaldehyde11. In addition, modified fixation formulations are increasingly emerging to maximize the retention of the original properties of the retina and the target cells9,10. Different retinal fixation treatments may affect the retinal structure, protein immunogenicity, fluorescence excitation , and attenuation quenching cycle differently12,13. Retinas fixed with Davidson's solution are more morphologically intact compared to those fixed with formalin, but Davidson's solution is less compatible with some antibodies, such as microglial marker-ionized calcium binding adaptor molecule 112. Considering the fragile nature of retinas, researchers would naturally wonder whether the retinal integrity, as well as the properties and morphology of the target cells, will change after long-term storage. However, the possible effects of fixation solution on retinal and RGC cell morphology after storage for several months have rarely been reported. The optimization of retinal fixation is critical for the evaluation and preservation of RGCs. 
We provide a detailed description of a reliable and technically straightforward method that we use for whole-mount murine retinal staining. Our method emphasizes the proper preparation and storage of retinas for RGC investigation, taking into account the need for the long-term storage of retinal tissue as well as specific aspects of fluorophore formation or degradation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well cell culture cluster</td>
			<td>Costar</td>
			<td></td>
			<td>Eyeball fixation</td>
		</tr>
		<tr>
			<td>24-well hemagglutination plate</td>
			<td>Labedit Company</td>
			<td></td>
			<td>Incubation antibody</td>
		</tr>
		<tr>
			<td>Adhesion microscope slides</td>
			<td>Citotest</td>
			<td></td>
			<td>Or similar</td>
		</tr>
		<tr>
			<td>Anti-fluorescent quenching mountant</td>
			<td>Servicebio</td>
			<td>G1401</td>
			<td>Slow down fluorescence quenching</td>
		</tr>
		<tr>
			<td>BSA (bovine serum albumin)</td>
			<td>Servicebio</td>
			<td>GC305010</td>
			<td>Blocking reagent</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>OLYMPUS</td>
			<td></td>
			<td>Apply 40x objective lens</td>
		</tr>
		<tr>
			<td>Curved scissors</td>
			<td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td>
			<td></td>
			<td>Dissecting tools</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>RWD Life science Co.,LTD&#160;</td>
			<td>77001S</td>
			<td>Dissecting tools</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td>
			<td></td>
			<td>Dissecting tools</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd.</td>
			<td>20210624</td>
			<td>GC&#8805;99.5%</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>SecheVite</td>
			<td></td>
			<td>Sealing agent</td>
		</tr>
		<tr>
			<td>Needles&#160;</td>
			<td>Shanghai Kindly Enterprises Development Group Co., Ltd.</td>
			<td></td>
			<td>Accelerate the fixation</td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution</td>
			<td>Servicebio</td>
			<td>G1101</td>
			<td>Eyeball fixation</td>
		</tr>
		<tr>
			<td>PBS (phosphate buffered saline pH 7.4)</td>
			<td>Servicebio</td>
			<td>G0002</td>
			<td>Rinse the eyeball&#160;</td>
		</tr>
		<tr>
			<td>Primary antibody: guinea pig anti-RNA-binding protein with multiple splicing (RBPMS)</td>
			<td>PhosphoSolutions</td>
			<td>Cat. #1832-RBPMS</td>
			<td>For immunofluorescence. Used at 1:400</td>
		</tr>
		<tr>
			<td>Secondary antibody: Cy3 affiniPure donkey anti-guinea pig IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>706-165-148</td>
			<td>For immunofluorescence. Used at 1:400</td>
		</tr>
		<tr>
			<td>Straight scissors</td>
			<td>Jiangsu Kanghua Medical Equipment Co., Ltd.</td>
			<td></td>
			<td>Dissecting tools</td>
		</tr>
	</tbody>
</table>

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.

After dissection, the retina should look like a flat four-leaf clover. In this study, by using the protocol outlined above, the retina turned white after methanol was added (Figure 1). Meanwhile, the retina changed from soft to pliable and flat. Next, the RGCs were labeled with anti-RBPMS8. Four image fields were taken in the whole-mount retina (n = 3) using a confocal microscope (eyepiece: 10x; objective: 40x). Representative views of visualized RGCs of retinas before methanol processing and after 12 months of long-term preservation in methanol, are demonstrated in Figure 2. The fluorescence intensity showed no significant changes. Methanol preservation did not affect the RGC immunostaining.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65222/65222fig01.jpg" /> 
Figure 1: Retinal changes after methanol treatment. (A) Before methanol treatment. (B) After methanol treatment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65222/65222fig01large.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/65222/65222fig02.jpg" /> 
Figure 2: Effect of methanol on retinal ganglion cell (RGC) immunostaining. (A) Retina without methanol treatment. (B) Retina with 12 month storage in methanol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65222/65222fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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.

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 ...

methanol-based-whole-mount-preparation-for-investigation-retinal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.1K Views.  Renmin Hospital of Wuhan University. 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.

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

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 ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

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

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Chapters in this video

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

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells