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

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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. 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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. 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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 investigations<su...

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><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&amp;nbsp;</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&amp;ge;99.5%</td></tr><tr><td>Nail polish</td><td>SecheVite</td><td></td><td>Sealing agent</td></tr><tr><td>Needles&amp;nbsp;</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&amp;nbsp;</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 befor...

Watch this Scientific Journal Video about Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells at JoVE.com

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 ...

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3.2K Views.  Renmin Hospital of Wuhan University. Het onderzoek is gericht op het bieden van een aangepaste retina-isolatieworkflow en een protocol voor de opslag van weefselmonsters, wat nuttig en praktisch is voor het onderzoeken van RGC's. Netvliezen zijn fragiel en vouwen, wat experimenteel onderzoek beperkt bij het maken van een retinale pleister met hele montage. Bovendien vereisen alle netvliezen geen onmiddellijke immunostain.Soms moeten extra netvliesmonsters worden bewaard voor verder gebruik. We bieden een gedetailleerde be...

Video: Auteur in de schijnwerpers: op methanol gebaseerd preparaat voor de hele montage voor het onderzoek van retinale ganglioncellen  

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 ...

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. ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

Biology

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

Developmental Biology

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 ...

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 ...

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 ...

Immunostaining and AI-Based Counting of RGCs in Mouse Whole-Mount Retina

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve as valuable animal models for studying the pathogenesis and treatment of glaucoma due to their relatively homogenous genetic background and retinal ganglion cells (RGCs), which structurally resemble those in humans. Accurate assessment of RGC damage and treatment outcomes in mouse glaucoma models requires determining the RGC number across the entire retina. This protocol outlines a comprehensive method involving the isolation of the whole retina, labeling RGCs with specific antibodies, and rapid, accurate automatic counting of RGCs using an AI-based program. The streamlined approach allows for efficient and precise quantification of RGC numbers in mouse retinas, facilitating the evaluation of RGC degeneration and potential therapeutic interventions. By enabling researchers to assess the extent of RGC damage, this protocol contributes to a deeper understanding of glaucoma pathogenesis and aids in developing effective treatment strategies to manage and prevent vision loss.

Author Spotlight: Efficient Retinal Ganglion Cell Counting in Mouse Models of Glaucoma for Treatment Evaluation

This protocol describes the procedure for isolating the whole-mount mouse retina and performing immunostaining to label all retinal ganglion cells (RGCs). The process is followed by imaging and automatically counting RGCs using AI-based software, providing a simple, fast, and accurate method for quantifying RGCs in the entire mouse retina.

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve ...

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

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