Name: Examination of Anatomical Features of Retinal Ganglion Cells Under N-methyl-D-aspartic Acid (NMDA)-induced Excitotoxicity (Video) | JoVE Journal
Uploaded: 2025-09-19T12:40:00.000+00:00
Duration: 7 min 11 s
Description: Learn to investigate retinal ganglion cell degeneration under NMDA-induced excitotoxicity using genetically directed alkaline phosphatase labeling in mouse retinas. This method reveals novel phenotypes in dying RGCs.

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5 minutes

This work was supported by grants from the National Institutes of Health-National Eye Institute to C.A.M. (EY024376) and in part by National Eye Institute Vision Core Grants P30EY028102 (UTHealth). We also acknowledge the financial support from the Stephen Lasher Endowment (UTHealth) and the Raye White Endowment (UTHealth).

All animal procedures followed the US Public Health Service Policy on Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at The University of Texas McGovern Medical School at Houston. The compositions of the solutions are listed in the Table of Materials.
1. Injection of tamoxifen
<ol>
	<li>Mix 30 mg of tamoxifen with 30 &#956;L of ethanol. In sub-boiling water, heat 1 mL of sunflower seed oil and the tamoxifen/ethanol mixture.</li>
	<li>After the tamoxifen has dissolved in the ethanol, quickly add 1 mL of heated sunflower seed oil to the tamoxifen solution. Mix well by pipetting.</li>
	<li>Mice of either sex at various ages between 2 to 6 months were used. Inject the tamoxifen solution at a dose of 100 &#956;g/g body weight intraperitoneally using an insulin syringe. Determine the number of tamoxifen injections based on the mouse line. In this protocol, tamoxifen was administered once daily for four consecutive days. 
	NOTE: Since the tamoxifen solution is mixed with dense oil, it is difficult to handle with a syringe that has a detachable needle. Thus, an insulin syringe with an attached needle is preferred for tamoxifen injection.</li>
	<li>House the mouse in groups of no more than 5 in individually ventilated cages. until the NMDA injection.</li>
</ol>
2. Injection of NMDA solution into the mouse eyes
<ol>
	<li>Prepare 20 mM of NMDA solution in sterile 1x PBS. Wash the Nano-injector with a 35G needle attached using 100% ethanol 20x, then wash with sterile ultrapure water an additional 20x. Fill the Nano-injector with 1 &#956;L of NMDA solution.</li>
	<li>Anesthetize the mouse by intraperitoneal injection of a ketamine/xylazine in 1X PBS (80/10 mg per kg body weight) solution mixture. Assess unconsciousness by reflex withdrawal response to tail and toe pinching.</li>
	<li>Place the anesthetized mouse on a stereomicroscope. Position the mouse with its right eye facing upward and its nose pointing toward the right side. Apply a drop of 0.5% proparacaine hydrochloride solution to the right eyeball for local anesthesia.</li>
	<li>Use a sterile 30G injection needle to puncture the limbus at the 7 o&#39;clock position, creating a small hole. Monitor the needle&#39;s location and direction under the stereomicroscope. Ensure the size of the injection needle is slightly larger than the nano-injection needle to allow easy insertion.</li>
	<li>Carefully remove the injection needle from the eyeball and dispose of it properly. Use the stereomicroscope for guidance, insert the nano-injection needle into the vitreous through the limbus at a 45&#176; angle.</li>
	<li>Slowly inject 1 &#956;L of NMDA solution into the vitreous. After the injection, keep the nano-injection needle in the vitreous for approximately 10 s to prevent the outflow of the injected solution.</li>
	<li>Carefully and slowly withdraw the nano-injection needle from the eye. Apply 0.5% erythromycin ophthalmic ointment to the cornea of the injected eye.</li>
	<li>Place the mouse in a cage on a heating pad. Monitor the mouse every 15 min until it has fully recovered from anesthesia. House the mouse for one to two weeks until tissue collection.</li>
</ol>
3. Tissue collection and Alkaline Phosphatase (AP) staining
<ol>
	<li>Euthanize the mouse with CO2&#160;asphyxiation, followed by cervical dislocation. Carefully cut the muscles connecting to the eyeball and the optic nerve with Vanna scissors.</li>
	<li>The eyeball was then picked up with forceps and transferred into separate labeled 2.0 mL tubes.</li>
	<li>Fix the eyeballs in 10% neutral buffered formalin for 10 min at room temperature. Flat mount the retinas onto a cellulose nitrate membrane filter as described below.
	<ol>
		<li>Transfer the eyeballs to 10 cm Petri dishes containing 1x PBS. Hold any muscle tissue attached to the eyeball using forceps. Create a hole at the limbus using a 30G injection needle. Insert the tip of Vanna scissors into the hole and cut around the ora serrata. Remove the lens from the eyecup using forceps.</li>
		<li>Make 3-4 cuts on the eyecup and use forceps to hold the RPE and gently remove the RPE from the retina.</li>
		<li>Cut a small piece of cellulose nitrate membrane filter and soak it in 1x PBS. Place the isolated retina onto the filter. Gently push a peripheral area of the retina with the tip of a fine paintbrush to attach the edge of the retina to the filter.</li>
		<li>Hold the edge of the filter and slowly pull it out of the 1x PBS, ensuring the retina remains attached and flattens onto the filter. Place the retina/filter on a paper towel for 1 min to help secure the attached retina to the filter.</li>
		<li>Transfer the retina/filter back to a petri dish with 1x PBS. Carefully remove any debris, hair, and vitreous from the retina using forceps and/or a fine paintbrush, as much as possible. To distinguish the injected and uninjected retinas, trim filters in different shapes.</li>
	</ol>
	</li>
	<li>Transfer the retina/filter back to a Petri dish. Fix the retina in 10% neutral buffered formalin for 5 min at room temperature with gentle agitation.</li>
	<li>Prepare a 65&#160;&#176;C water bath. Preheat 30 mL of 1x PBS in a 50 mL glass beaker. Transfer the retina/filters directly into the preheated 1x PBS. Incubate retinas at 65&#176;C for 30 min. 
	NOTE: The heat treatment inactivates the endogenous AP activity, which causes background staining in the retina. It is crucial to treat the retina in the heated solution to effectively eliminate this background.</li>
	<li>After heat treatment, transfer the retinas to a 30 mm Petri dish containing AP buffer (100 mM Tris, pH 9.5, 100 mM NaCl, 50 mM MgCl2).</li>
	<li>Incubate the retinas in the AP buffer at room temperature for 5 min with gentle agitation. 
	NOTE: This step exchanges the buffer in the retina from 1x PBS to AP buffer, preventing chemical precipitation during color development.</li>
	<li>Replace the AP buffer with staining solution (30 &#956;L of 10 mg/mL BCIP and 60 &#956;L of 10 mg/mL NBP in 10 mL of AP buffer).</li>
	<li>Place the Petri dish in a light-excluding box. Develop the AP reaction at room temperature for 1 to 2 days with gentle agitation until the intensity of the AP staining is satisfactory for imaging.</li>
	<li>Stop the reaction by washing the retinas with 1x PBS for 5 min, 2x, at room temperature with gentle agitation.</li>
	<li>Postfix the retinas with 10% neutral buffered formalin for 10 min at room temperature with gentle agitation.</li>
	<li>Wash the retinas with 1x PBS for 5 min, 3x, at room temperature with gentle agitation. Dehydrate the retinas by incubating them in 30% ethanol, followed by 50% ethanol for 30 min each at room temperature with gentle agitation.</li>
	<li>Use a fine paintbrush to gently remove the retinas from the filter paper. Dehydrate the filter-free retinas in 70% ethanol for 30 min, followed by two 30-min incubations in 100% ethanol at room temperature with gentle agitation.</li>
	<li>Clear the retinas in a glass Petri dish with a benzyl benzate/benzyl alcohol (2:1, v/v) clearing solution for 30 min to 2 h at room temperature with gentle agitation. 
	NOTE: This clearing procedure should not be performed overnight, as prolonged incubation can often result in the loss of AP staining. Clearing solution can melt plastic, so the procedure should be performed in a glass container.</li>
	<li>To mount the retinas onto a glass slide, apply double-sided tape to the slide to create a platform for the coverslip and retina. Place the retinas on the slide, fill the space with clearing solution, apply the coverslip, and seal the slide with nail polish to secure the retina and coverslip.</li>
	<li>Image the AP-stained retinas using a Zeiss Apotome microscope.</li>
</ol>

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<li>Badea, T. C., Wang, Y., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+noninvasive+genetic%2Fpharmacologic+strategy+for+visualizing+cell+morphology+and+clonal+relationships+in+the+mouse%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">A noninvasive genetic/pharmacologic strategy for visualizing cell morphology and clonal relationships in the mouse.</a> J Neurosci. 23 (6), 2314-2322 (2003).</li>
<li>Mao, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eomesodermin%2C+a+target+gene+of+Pou4f2%2C+is+required+for+retinal+ganglion+cell+and+optic+nerve+development+in+the+mouse%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Eomesodermin, a target gene of Pou4f2, is required for retinal ganglion cell and optic nerve development in the mouse.</a> Development. 135 (2), 271-280 (2008).</li>
<li>Shi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+interactions+between+Brn3+transcription+factors+in+retinal+ganglion+cell+type+specification%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Genetic interactions between Brn3 transcription factors in retinal ganglion cell type specification.</a> PLoS One. 8 (10), e76347(2013).</li>
<li>Muzyka, V. V., Brooks, M., Badea, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Postnatal+developmental+dynamics+of+cell+type+specification+genes+in+Brn3a%2FPou4f1+Retinal+Ganglion+Cells%5BTitle%5D)+AND+%22Neural+Dev%22%5BJournal%5D)">Postnatal developmental dynamics of cell type specification genes in Brn3a/Pou4f1 Retinal Ganglion Cells.</a> Neural Dev. 13 (1), 15(2018).</li>
<li>Liu, S., Aldinger, K. A., Cheng, C. V., Kiyama, T., Dave, M., McNamara, H. K., Zhao, W., Stafford, J. M., Descostes, N., Lee, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NRF1+association+with+AUTS2-Polycomb+mediates+specific+gene+activation+in+the+brain%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">NRF1 association with AUTS2-Polycomb mediates specific gene activation in the brain.</a> Mol Cell. 81 (22), 4663-4676.e8 (2021).</li>
<li>Kiyama, T., Altay, H. Y., Badea, T. C., Mao, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pou4f1-Tbr1+transcriptional+cascade+controls+the+formation+of+Jam2-expressing+retinal+ganglion+cells%5BTitle%5D)+AND+%22Front+Ophthalmol%22%5BJournal%5D)">Pou4f1-Tbr1 transcriptional cascade controls the formation of Jam2-expressing retinal ganglion cells.</a> Front Ophthalmol. 3, 1175568(2023).</li>
<li>Park, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Promoting+axon+regeneration+in+the+adult+CNS+by+modulation+of+the+PTEN%2FmTOR+pathway%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway.</a> Science. 322 (5903), 963-966 (2008).</li>
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The authors declare no competing interests.

In this study, we utilized a genetically directed sparse AP labeling system in conjunction with a number of genetically manipulated mouse lines to examine NMDA-induced degenerative events in various RGC and AC subtypes and uncovered several hallmark phenotypes29,31,39. The most noticeable phenotype is the significant reduction of RGCs and ACs in the NMDA-injected retinas (Figure 2A-D</strong...

Traumatic brain injury (TBI) is a sudden injury that causes neuronal damage to the brain. In severe forms of TBI, such as those caused by car accidents or sports collisions and blast injuries due to explosions, mechanical forces may disrupt cell architecture, leading to inflammation, oxidative stress, excitotoxicity, and eventually neuronal death1,2.
Retina, the most accessible part of the central nervous system (CNS), has served as a model TBI system to study the molecular mechanisms underlying excitotoxicity and to develop strategies for therapeutic intervention3,4,5. The most pertinent retina models for TBI studies are the optic nerve crush model and N-methyl-D-aspartic acid (NMDA)-induced excitotoxicity of retinas. In the NMDA-induced excitotoxicity retina model, retinal ganglion cells (RGCs) were identified as the most susceptible type of retinal neurons because RGCs utilize glutamate as a major excitatory neurotransmitter and various types of glutamate-receptors, including AMPA receptors (AMPARs), KAPA receptors (KAPARs), and NMDA receptors, to trigger axon potential6. In addition to RGCs, other retinal cells and neural circuitries could also be affected by NMDA treatment7,8,9. Intra-vitreous application of NMDA elicits prolonged opening of the NMDA-receptor, which leads to an excess influx of calcium into cells, causing over-excitatory neuronal activity and imbalanced calcium homeostasis10,11,12,13.
The NMDA receptor is a heteromeric complex composed of three subunits: GluN1, GluN2, and GluN314,15,16,17,18. GluN1, encoded by Grin1, contains eight isoforms in mice due to alternative splicing of Grin1. There are four different GluN2 subunits (a-d), which are encoded by four separate genes: Grin2a-2d. There are two different GluN3 subunits (a and b), which are encoded by Grin3a and 3b. According to scRNA-seq data19,20, Grin1 is highly expressed in most RGC subtypes (Figure 1A) as well as a few amacrine cell (AC) subtypes with weak to moderate expression levels (Figure 1B). Grin2a is enriched in fewer RGC and AC subtypes, whereas Grin2b is expressed moderately in most RGC and many AC subtypes (Figure 1A,B). Expression levels of Grin2c, Grin2d, and Grin3b in RGCs and ACs are extremely low (Figure 1A,B). Grin3a expression can be seen in a few RGC subtypes and weakly in a few ACs (Figure 1A,B). In addition to the role of NMDA receptors, calcium influx-triggered excitotoxicity through calcium-permeable AMPARs has been described in multiple disease models21. The expression of NMDA receptor subunits may provide clues as to why NMDA-induced excitotoxicity targets mainly RGCs as well as ACs9,10,14,22,23.
Conventional methods of assessing gross NMDA-induced retinal phenotypes include hematoxylin and eosin (H&#38;E) staining to detect histological abnormalities, TUNEL assay for cell death, and immunofluorescent (IF) staining using a cell-type-specific biomarker8,10,24,25,26. At the cellular level, confocal imaging and dye-filling techniques have been used to investigate the sequence of events in dying RGCs in the NMDA-induced retinal model. These methods have provided valuable insights into the subcellular compartments of the dying RGCs and allowed for the identification of the types of resilient RGCs that survive under NMDA insult27,28.
Here, we employ genetically activated alkaline phosphatase (AP) staining to investigate the anatomical features and phenotypes of mouse retinal neurons under NMDA-induced excitotoxicity29,30,31. The rationale for selecting this method is due to its simplicity, cost-effectiveness, and ability to obtain a comprehensive overview of the anatomical features of neuronal subtypes across the entire retina, making it well-suited for gross assessments and large-scale comparative analyses. This approach provides novel insights into how distinct, genetically marked retinal neurons respond to NMDA at both gross and subcellular levels. We anticipate that this strategy will be broadly applicable for assessing morphological changes in various disease models that affect neuronal architecture and synaptic organization4,26,32,33.

<table><tbody><tr><td>0.5% erythromycin ophthalmic ointment&amp;nbsp;</td><td>Bausch+Lomb</td><td>24208-910-55</td><td></td></tr><tr><td>0.5% proparacaine hydrochloride&amp;nbsp;</td><td>Bausch+Lomb</td><td>24208-730-06</td><td></td></tr><tr><td>10% neutral buffered formalin&amp;nbsp;</td><td>VWR</td><td>89370-094</td><td></td></tr><tr><td>10 cm petridish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>10x PBS</td><td>Fisher Scientific</td><td>BP399</td><td>Dilute in water to create 1x PBS and autoclave.&amp;nbsp;</td></tr><tr><td>2.0 ml tube</td><td>VWR</td><td>525-1136</td><td></td></tr><tr><td>30 gauge injection needle</td><td>EXELINT International</td><td>26437</td><td></td></tr><tr><td>30 mm petridish</td><td>Falcon</td><td>35208</td><td></td></tr><tr><td>Benzyl alchohol&amp;nbsp;</td><td>Acros Organics</td><td>447000010</td><td></td></tr><tr><td>Benzyl benzate</td><td>Acros Organics</td><td>105860010</td><td></td></tr><tr><td>Cellulose nitrate membrane filter</td><td>Cytiva</td><td>7188-002</td><td></td></tr><tr><td>Cover slip</td><td>Electron Microscopy Sciences</td><td>72204-01</td><td></td></tr><tr><td>Double sided tape</td><td>Scotch</td><td>597020</td><td></td></tr><tr><td>Ethanol</td><td>Decon Labs</td><td>2716</td><td>Avoid Fisher Bioreagents (Cat#: BP2818) for dehydtration after AP staining. This reagent distorts retinas.</td></tr><tr><td>Forceps</td><td>TED PELLA</td><td>505</td><td></td></tr><tr><td>Heating pad</td><td>Theratherm</td><td>710298-001</td><td></td></tr><tr><td>Insulin syringe</td><td>BD</td><td>329424</td><td></td></tr><tr><td>Ketamine</td><td>Dechra</td><td>17033010110</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Axio Imager 2</td><td></td></tr><tr><td>Nail polish</td><td>Electron Microscopy Sciences</td><td>72180</td><td></td></tr><tr><td>Nanofil injector</td><td>World Precision Instruments</td><td>NANOFIL</td><td></td></tr><tr><td>Nanofil needle</td><td>World Precision Instruments</td><td>NF25BL</td><td></td></tr><tr><td>NBT/BICP reagent kit</td><td>Invitrogen</td><td>N6547</td><td></td></tr><tr><td>NMDA</td><td>Sigma-Aldrich</td><td>M3262</td><td></td></tr><tr><td>Paint brush</td><td>Kingart</td><td>6650</td><td></td></tr><tr><td>Slide glass</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Stereo microscope</td><td>Motic</td><td>DM143</td><td></td></tr><tr><td>Sunflower seed oil</td><td>Sigma-Aldrich</td><td>S5007</td><td></td></tr><tr><td>Tamoxifen</td><td>Sigma-Aldrich</td><td>T5648</td><td>Store tamoxifen solution at 4 &amp;deg;C in a light-tight box. Use tamoxifen solution within one week.</td></tr><tr><td>Vanna scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>Waterbath</td><td>Lab-Line</td><td>18052</td><td></td></tr><tr><td>Xylazine</td><td>Dechra</td><td>17033-0099-05</td><td>Product name: Rompun</td></tr></tbody></table>

examination of anatomical features of retinal ganglion cells under n-methyl-d-aspartic acid (nmda)-induced excitotoxicity

Examination of Anatomical Features of Retinal Ganglion Cells Under N-methyl-D-aspartic Acid (NMDA)-induced Excitotoxicity

Retinal ganglion cells (RGCs) are the output neurons of the retina, responsible for transmitting visual information to the brain. In the mature mouse retina, over 40 RGC types have been identified based on genomic, morphological, and functional characteristics. RGC degeneration is a hallmark of various debilitating retinal diseases, including glaucoma and traumatic optic neuropathy. Gaining deeper insight into the cellular and molecular mechanisms underlying RGC degeneration, survival, and resilience across different disease conditions is crucial for developing effective therapeutic interventions.&#160;To investigate RGC degeneration, we adopted a simple yet powerful technique previously used extensively in retinal development studies for RGC morphologies and central projection. Here, we apply this method to assess the anatomical phenotypes of RGCs under NMDA-induced excitotoxicity. This approach enables the visualization of large numbers of degenerating RGCs over time, allowing us to map the sequence of degenerative events, identify hallmark phenotypes of dying RGCs, and distinguish RGCs that are resistant to NMDA-induced damage.

NMDA application in the vitreous induces RGC death, which has conventionally been detected using histological staining or immunofluorescence (IF) staining with RGC markers. In our experimental setting, by using the conventional IF strategy with a pan-RGC marker RBPMS, we confirmed that the number of RGCs decreased by approximately 65%, 1 week after NMDA injection (Figure 2E-G). Previous studies have discovered that Tbr2-expressing RGCs are indeed ipRGCs34</...

Here, we describe the use of a genetically directed alkaline phosphatase (AP) labeling method to investigate the anatomical features and phenotypes of mouse retinas under NMDA-induced excitotoxicity. In conjunction with several retinal ganglion cell (RGC) subtype-specific reporter lines, we uncover novel phenotypes in dying RGCs throughout the degenerative process.

Retinal ganglion cells (RGCs) are the output neurons of the retina, responsible for transmitting visual information to the brain. In the mature mouse ...

We are trying to understand the development, survival, and the degeneration of retinal ganglion cell using remodel.We are using genetically modified mouse line to label specific cell types to monitor the degeneration of these cells.With this protocol, we can investigate anatomical features and phenotypes of retinal ganglion cells under NMDA-induced excitotoxicity.This approach provides new insights into how retinal ganglion cells respond to NMDA at gross and subcellular levels.To begin, obtain the eyeball from the NMDA-injected mouse after euthanization, and transfer it into a separate labeled two milliliter tube.Fix the eyeball in 10%neutral buffered formalin for 10 minutes at room temperature, and obtain a cellulose nitrate membrane filter to flat mount the retina.After fixation, transfer the eyeball to a 10-centimeter Petri dish containing 1

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Intravitreous injection is a widely used technique in visual sciences research for ocular diseases or as direct application of local treatment. This video demonstrated a protocol for intravitreous injection using a 1ml syringe with glass pipette. Useful tips about avoiding massive bleeding and lens damage are given.

Intravitreous Injection for Establishing Ocular Diseases Model

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Morphometric Analyses of Retinal Sections

Source: Lani-Louzada, R., et al.  Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022).This video demonstrates the quantification of retinal ganglion cells from chemically fixed rat retinas subjected to full-circle cauterization of the limbal vascular plexus. The process includes permeabilization, antibody staining, and nuclear counterstaining, followed by visualization under an epifluorescence microscope to assess the impact of...

Quantification of Retinal Ganglion Cell Somas Following Cauterization-Induced Ocular Hypertension 

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.

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

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the naïve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

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

Learn to investigate retinal ganglion cell degeneration under NMDA-induced excitotoxicity using genetically directed alkaline phosphatase labeling in mouse retinas. This method reveals novel phenotypes in dying RGCs.

Examination of Anatomical Features of Retinal Ganglion Cells Under N-methyl-D-aspartic Acid (NMDA)-induced Excitotoxicity

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