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Retinal Ganglion Cells - Science topic

Retinal Ganglion Cells are neurons of the innermost layer of the retina, the internal plexiform layer. They are of variable sizes and shapes, and their axons project via the OPTIC NERVE to the brain. A small subset of these cells act as photoreceptors with projections to the SUPRACHIASMATIC NUCLEUS, the center for regulating CIRCADIAN RHYTHM.
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I reprogramming glia in vitro into retinal ganglion cells. When I stain the induced neuron with retinal ganglion cells marker (Rbpms), I have a weak signal in nuclei, however, Rbpms should be in the cytosol. I check the control (only the 2nd antibody or Igg) but I did not see the same signal. The other marker of retinal ganglion cells (Brn3b) doesn't work and I am ordering from another producer.
Anyone experienced the same phenomenon and could give me an advice?
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Sergei Gaidin Thank you so much for your great answer. I will discuss this with my labmates carefully and try some modifications. Again, thank you.
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is there anyone has ever used glutamate to induce primary retinal ganglion cells death? what concentration do you use? or on neuronal PC12 cells? or other neuronal cells?
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We have used 2.5 mM and 10 mM in PC12 cells but I won't use PC12 cells as they are passe' (it's hard to publish). Best. Adriana
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I've been doing CTB-Alexa injections for tracing from the eyes to the dLGN, and they have been working perfectly. I'd like to do intraocular injections of WGA-Alexa to trace from the dLGN to the visual cortex but haven't had much success to far. Does anyone have any experience with these injections and the most effective concentration of the WGA-Alexa dye to use? I think I may have been using too high a concentration so far.Thanks
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Jeremy Petravicz Have you been using them in adults for eye to dLGN connections?
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I have been trying to record neuronal (ganglion cell) activity from the chick retina using a MEA 1060-BC-INV amplifier and a 60 channel MEA. The signal to noise ratio is low, and thus is hindering further analysis like spike sorting. Is there any tips for improving such recordings, and also is it necessary to have a perfusion setup for stable recordings, or can I manually change the medium periodically over the course of a recording? Will this affect the recording adversely with regard to there being too much disturbance during medium change?
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MEA preamplifier stages are usually placed in close proximity to the neuronal cell culture coupled to the MEA substrate to minimize signal attenuation and noise coupling, enhancing the signal-to-noise ratio (SNR) of recordings. This raises the need to limit the amount of produced heat by the circuitry surrounding the array, in order to prevent significant cell culture temperature upward drifts able to perturb neuronal physiology and cell viability (i.e., >38°C) . This issue requires attention mainly in experimental setups integrating climate control capabilities (e.g., portable culturing and recording chamber or cell incubators embedding MEA equipment) to maintain cells viability during prolonged MEA recordings (i.e., >1 hour). Indeed, the encapsulation of the MEA recording equipment in a confined space kept at physiological temperature hinders or slows down thermal dissipation. A common solution to perform climate-controlled recordings is to insert a commercial MEA preamplifier stage (i.e., the MEA1060 device sold by Multi Channel Systems GmbH) inside a cell incubator . However, the power consumption of the MEA1060 (i.e., 2 W) requires the integration of additional devices (e.g., heat sink) in order to neither damage cells due to overheating nor perturb the incubator temperature controller, raising possible issues of sterility and encumbrance. Besides the problem of overheating, the performance of MEA equipment integrated in such setups is downgraded by the high level of humidity (i.e., relative humidity > 90%) traditionally used in cell culture environments to maintain osmolarity and thus cell viability. This imposes to lower the humidity to ambient levels (i.e., <60%) in order not to damage MEA interface boards, which however induces a faster osmolarity increase
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I have sections of mouse retina that I'm staining with RBPMS and GFAP using a very standard Immunofluorescence protocol. GFAP positivity seems to consistently come up in some RGCs. This doesn't make sense. I've tried switching to TBS for the buffer, increasing the blocking incubation time (2 hrs), and using detergents (Triton (0.1%) and Tween (0.2%)).
Any thoughts? Why might this be happening?
Block: 10% Goat Serum, 1% BSA in PBS. 2 hours at RT
Primary: 1% GS, 1% BSA, primary Abs in PBS. O/N at 4*C.
Wash: 3x5min PBS
Secondary: 1% GS, 1% BSA, secondary Abs in PBS. 90 min at RT, protected from light.
Wash: 5min PBS, 10min PBS+DAPI, 5min PBS
Mounting: Antifade Gold
Primaries: GFAP (mouse, 1:100) and RBPMS (Guinea Pig, 1:100)
Secondaries: Goat anti-mouse (555 nm, 1:1000) and Goat anti-guinea pig (647 nm, 1:1000)
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Yes, this does look like a legitimate positive signal. I think it's either antibody cross-reactivity with another protein. Check the antibody quality (who purchased, when, what kind of antibody this is, to what region/sequence), if this antibody has been validated and used in your lab for a long time without problems, then it's a legitimate signal and there in fact are GFAP positive cells there in that location.
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I used to patch RGCs in a transgenic mouse line and defined 8 cell types (PV-0 to PV-7). Several types were electrically coupled, as revealed by neurobiotin the patch pipette (see attached figure: "PV cells"). I no longer work directly on retina, but I'm still fascinated by this feature of RGCs.
PV-7 has an asymmetric dendritic field (but only weak direction selectivity when the stimulus is centred on receptive field centre). The Sanes group named them JAM-B cells. I observed small electrically-coupled neurons within the dendritic field of PV-7 neurons close toi the GCL (see attached figure: "DS and coupling"). I'm curious if it is now known what these coupled cells are and what they would contribute to visual responses?
I am following your project with great interest!
Also see Fig S1b in Farrow et al Neuron 2013
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Hi Tim,
Thanks for the question!
Well this cell (and Josh's JAM-B cells also) looks like the one I name to G15 in my characterisation (Volgyi et al. 2009; and it was C6 in Sun et al 2002). I always foun this cell coupled to a cohort of regularly and closely spaced somata in the INL. We concluded that they must have been amacrine cells. I have never seen these cells tracer coupled to either other G15 or displaced amacrine cells though.So, to answer your question I'm not sure what those cell bodies could be in the GCL. However, their shape and size are very similar to your NB injected cell, which is an indication towards homologous coupling. I'll check the literature what kinda coupling patterns have been describend JAM-B cells in the literature and get back to you.
..in case I forget.. please drop me a reminder message!
best,
Béla
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Cones and IpRGCs have a overlap in spectral sensitivity , what is the best method to assess IpRGCs response alone without cones response in a experimental setting whie performing EEG.
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Hi
In the second link I provided, it is possible to isolate these cells by inhibiting rods and cones response using a drug injection. Then , you can perform EEG in this setting. You need approval to try it on mice etc....
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I've used Pubmed to look up my gene or protein of interest in RGCs and have come up with nothing. Additionally, my attempt to use the Allen Brain Atlas proved unfruitful.
Does anyone know of a user-friendly site to look up expression profiles in mouse RGCs?
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If you are looking to investigate the expression profile yourself, I suggest a microarray analysis or RNAseq.
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I'm interested in locating the myelin transition zone in serial cross sections of the mouse optic nerve. What antibody would you recommend for use in lightly fixed cryostat sections?
thanks in advance!
Mary Ellen
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RGC axons are nicely detected with anti-pNFH antibody (Rt-97 clone)
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The ability to detect photons near the sensory threshold will probably be compromised by the fact that the organism/retina also emits electromagnetic waves (e.g. as heat). Hecht et al (1942) used a dark adaptation of only 30 min, which still permitted light sensitivity - so the retina had probably not reached equilibrium with the environment. Dodt and Echte (1961) used a dark adaptation of 4 hours, still without the dark adapted retina ever reaching equilibrium with the environment. I had used Dodt's original setup in his lab at Bad Nauheim and retinal ganglion cells seemed sensitive even after 12 hours of dark adaptation. Are there any more enlightening estimates on the visual threshold (irradiance, photon flux, etc) below which the dark-adapted mammalian retina would not be sensitive to visible light because of environmental and self-induced photon noise? This should be near the thermal noise of the retina in the visible range. So a different way to phrase the question would be "what is the thermal noise level of the retina in the visible wavelengths?" ideally with the minimal realistic photon flux of the environment taken also into consideration.
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Dear Nicolaus & Tony,
Look, please, interesting information from the site
It is in Russian, so I translated into  English some sentences connected the problem raised -
In 1941, researchers from Columbia University conducted an experiment: subjects were brought into a dark room and give their eyes some time to adapt. To achieve the full sensitivity of the sticks takes a few minutes: that's why, when we turn off the room light, then at some time lose the ability to ever see.
Then, in the face of the subjects were sent a flashing blue-green light. With a probability of higher than normal accident participants recorded a flash of light when hit on the retina 54 of photons.
Not all the photons reaching the retina, the photosensitive cells are recorded. Given this fact, the researchers came to the conclusion that only five of photons that activate five different sticks in the retina, enough people saw the flash.
Although the vision most of us are limited visible light spectrum, and people with aphakia — absence of the eye lens (as a result of surgery for cataract or sometimes due to a birth defect) is able to see ultraviolet waves.
In a healthy eye the lens blocks the UV wave range, but in its absence, man is able to perceive wavelengths of about 300 nanometers as blue and white color.
The study 2014 it is noted that in some sense we can all see and infrared photons. If two photons almost simultaneously fall on the same retinal cell, their energy can be summed, turning invisible wavelengths, for example, in 1000 nanometers in the visible wave length of 500 nanometers (the majority of us perceives this wave length like the cool green color).
I have to do with a very small objects - ciliated protists - so, was interesting for me how large infusoria I can see! 30-40 microns but colourless and when moving. May be tetrachromates can see even such small in colour?
Andrey
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I'm looking for H&E stained slides of retina to find how does the retina look at different stages of retinal ganglion cell death in diseases like glaucoma. Haven't found much on google.
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"Role of hypoxia-inducible factor-1α in preconditioning-induced protection of retinal ganglion cells in glaucoma" from Zhu et al. may help you.
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I am trying to use a Nissl/Cresyl Violet stain to visualize ganglion cells in flat-mounted bird retinas. However, I don't think I'm seeing any individual cells after staining - all I've got is fairly uniform purple/pink retinal tissue. I believe that at the magnification I'm looking (200x) the cells should be visible. I'm new to these techniques and would really appreciate any input.
The retinas have been fixed in 4% PFA and stored in PB buffer until dissection. They're bleached in hydrogen peroxide and mounted on gelatin-subbed slides. My staining procedure is:
Xylene 10 min
100% ethanol 3 min
90% ethanol 2 min
70% ethanol 2 min
20% ethanol 2 min
ddH2O 2 min
Cresyl Violet 20 min
ddH2O brief rinse
20% ethanol 30 sec
70% ethanol 30 sec
90% ethanol 30 sec
Differentiation Solution (900:1 95% ethanol & glacial acetic acid) 1 min
100% ethanol 3 min
Xylene 4 min
I'm attaching a photo taken at 200x magnification. The scale bar is not accurate. Any help would be much appreciated, thanks!
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I would recommend you touse specific RGC markers such as RBPMS or Brn3a 
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I'm looking into options for how to purify retinal ganglion cells, and recently came across neural tracers. It seems as though the preferred method is to inject into the brain, although some studies use postmortem tissue. Does anyone have experience with using the optic nerve from enucleated eyes to stain the retinal ganglion cells? I am hoping to FACS purify and then do RNA/DNA analysis. 
Is the time required for staining of the ganglion cells from the optic nerve going to be prohibatively long, or long enough to justify delivering an injection or dye sponge to the areas of interest in the brain? it seems most studies leave the crystal or dye for at least a week, is there a way to enhance the diffusion and marking of the cells?
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there are fluoro labelled biotins available, ...fluororuby should give good labeling after  a relatively short time...others have cut the optic nerve and placed a cuff soaked in the tracer on the end of the cut stump..there should be several articles using this if you do a pubmed search on RGC classification or retinal regeneration
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I am looking for a current clamp recording from starburst amacrine cells of the vertebrates' retina. I have already found a similar study (Figure1, A Unique Role for Kv3 Voltage-Gated Potassium Channels in Starburst Amacrine Cell Signaling in Mouse Retina - Ozaita et al) and I am looking for another one to cross-validate the findings.
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see miller and bloomfield PNAS 1983
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Ganglion cells in the retina have relatively small receptive fields (RF) (~1deg or less). If we assume that these neurons work as spatial filters for local contrast, we should expect that spatial frequencies (SF) stimuli lower than ~0.5cyc/deg would be hardly detected by these neurons because the local contrast within their RF is very small. However, signals with SF lower than 0.5cyc/deg are encoded by the retinal output and made accessible to higher level visual areas with wider RF (the neurons in these areas could therefore detect these kind of stimuli).
How are low spatial frequency signals transmitted then?
The simplest way would be that ganglion cells would respond not only to local contrast but also to local luminance. In this way low SF signals would be encoded in the ganglion cells output as a "place code". In other words every ganglion cell output could represent a "pixel" of the un-filtered luminance pattern projected on the retina preserving the low SF information.
Could you please suggest any sort of material addressing this question?
Thank you!
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Dear Giacomo,
I have attached one of our papers on quantitative histological estimates of RGC receptive fields in the human retina. I hope you will find it useful.
Zoran
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Currently, I use CD11b (abcam) and CD90 (BD) antibodies to purification of retinal ganglion cells by immunopanning. But I think the purification rate is low. Could you give me suggestion or information about the primary antibodies used in immunopanning.
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 May I ask what the antibody you use to remove glia cell. I also CD90. May I ask how about your purification rate. Thank you .
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I need help with a Retinal Ganglion Cell culture.
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In this paper we described the isolation and culture of embryonyl chick RGCs: Brocco M. A. and Panzetta P.: Survival and differentiation of purified embryonic chick retinal ganglion cells cultured at low density in a chemically defined medium. J. Neurosci. Meth. 75: 15-20. 1997
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Since the beginning of the 21st century, we know it exists other photo-receptors in the retina like the ipRGCs. These cells are not only implicated in the regulation of circadian rhythm but also in the contrast/motions detection. This diversity of functions can be explain by the different types of ipRGCs. It exist 5 types of ipRGCs (from M1 to M5) which have been characterized by their electrophysiological profile. Does anyone know if it exist specific molecular markers to differentiate these five ipRGCs types ?
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Dear Bastien,
Great question, but as far as I know there are specific markers for each type.
best wishes, Refik
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I'm aware that inhibitory synapses are GABA and glycine and excitatory are glutamate. Gephyrin looks like a reasonable option for inhibitory - i.e. stains glycine and some GABA. Wondering if PSD-95 is genuinely excitatory specific?
Definitely could do with a protocol for these kinds of antibodies in 10-12 micron retinal sections if anyone could point me towards good articles or other resources.
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I use the standard fixation we perfused the animal with 4%PFA for about 10 minutes.  Then incubate it overnight with 4%PFA . Followed by incubating the brains in 20% sucrose. 
If you have more questions I will be happy to help. 
best
Tahani
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I usually patch bipolar and amacrine cells in retinal slices, but I am trying to do some wholemount stuff at the moment. I have varying degrees of success with either clearing the membrane with an empty, broken pipette first, or just applying a lot of positive pressure on the recording pipette, punching through, and then recording. The latter technique seem to work reasonably well but if I have neurobiotin in the pipette it labels all the surrounding Muller cells. I know everyone does this differently, so I'd really like to hear different suggestions.
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A big question for RGC patchers. Not sure if you are still struggling with this but thought I would contribute any others who are struggling. I've tried collagenase, which works like a treat to dissolve the ILM and hyaluronidase on its own and in combo with collagenase. Either way you deliver the hyaluronidase, I found it to be too harsh for the retina and compromised integrity of the flatmount. Indeed, this is the issue with collagenase and hyaluronidase - do they impact on the patching data?
Because of this, I stick to using an empty patch pipette to "rip" through the ILM. This is a technique that does require practice - from "catching a wave" where you catch the ILM with the pipette tip and move forward, to then "ripping" a hole in the ILM by lifting the pipette slightly and then moving from side to side, eventually finishing by going so far to the side one way or the other that the pipette breaks its contact from the ILM (can also move back and to the side to achieve this). Then you hope that the ILM doesn't float back over to obscure the hole you've just created.
Having said all this, actually being able to do this "ripping" is very much reliant on the quality of your retinal dissection. Leaving any vitreous behind will result in a pipette that glides along the surface of the retina without catching any ILM. The presence of vitreous seems random to me as my dissection technique has become highly consistent but with varying degrees of vitreous left depending on the retina. But all the nuances of retinal dissection is probably best left for another thread...
Hope this helps.
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To support my in vitro experimental data, I start to try retina explant culture to check the axonal outgrowth of retina ganglia cell. I have tried many times with WT E18 mouse. The isolation of retina is good, and were cut into pieces round 200~500um. retina was put on the glass surface coated with laminin- (40 µg/ml) and poly-D-lysine- (100 µg/ml). The maintenance medium is containing Neural Basal medium and 1% N2 neuronal supplement (Gibco BRL), 1% penicillin-streptomycin (Gibco BRL), and 5 µg/ml insulin, 100 µg/ml transferrin, 20 nM progesterone, 0.1 mM putrescine, 30 nM selenium, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.4 mM glutamine. The problem is that the outgrowth rate of axon is very very low ( round 200~300 um round DIV10) compared to the previous report. some report said after 24h there is quite obvious axon out. some literatures used the collagen as matrix. some literatures just used glass coverslip as growth surface. I tried DRG explant culture with the same coating condition and medium condition, and the result was pretty good.
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Our key has always been to incubate the ex vivo retina at the air-water interface in the tissue culture dish.  So we have never put the retina directly on glass but rather on a transwell membrane.  The retina is grown photoreceptor side down.  Attached is a recent publication which gives most of the details of our methods. 
We have not assayed RGC axon growth - the Bonhoeffer stripe assay was developed specifically for this purpose - there is a recent review of it - Knöll et al Nature Protocols 2, - 1216 - 1224 (2007)  Published online: 10 May 2007 | doi:10.1038/nprot.2007.157 
Hope some of this helps - 
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I'm interested in immuno-labelling ganglion cells in mouse retina. I am mostly interested in antibodies that would also stain the dendrites (inner plexiform layer), but it's fine if it also labels the soma and axons. I see from the literature that Thy1 is often used, but I have struggled to get any of these antibodies to work in mouse (some work in rat).  
Does anyone have the product info for Thy1 or other antibodies that work for this purpose in mouse?
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MAP-1 and MAP-2 are excellent retina; ganglion cell-specific markers that will selectively label the dendrites in the IPL (cell body labeling is weaker). MAP-1 will preferentially label large ganglion cells and their dendrites; MAP-2 will preferentially label small ganglion cells and their dendrites. We use the old Huber and Matus  mouse monoclonals HM-1 and HM-2 (directed against MAP-1 and MAP-2 respectively) that are available commercially from several suppliers. We got our last batches from Sigma, who I think also have rabbit polyclonals against these antigens, if you need them in a non-mouse species.
Description of the antibodies: Huber G, Matus A. Differences in the cellular distributions of two microtubule-associated proteins, MAP1 and MAP2, in rat brain. J Neurosci 1984; 4:151-60.
Description of MAP distribution in the retina:   Tucker RP, Matus AI. Microtubule-associated proteins characteristic of embryonic brain are found in the adult mammalian retina. Dev Biol. 1988 Dec;130(2):423-34.
Brn3 is good only for somatic labeling of retinal ganglion cells.
Good luck.
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In whole mount preparations of the retina around postnatal day 6 appeared a layer of hyaloids vessel/inner limiting membrane (I guess this layer is it?) preventing the access to retinal ganglion cells (RGCs) for patch clamp experiments, only some RGCs are patchable. In later developmental stages this problem rises. The same problem appeared in Ca2+-imaging experiments, when retinae/RGCs are loaded with Fura2, this hyaloid vessel layer/inner limiting membrane prevents the loading of RGCs with the dye. How is it possible to avoid this problem? How to remove this layer without destroying the retina to get an good access to RGCs in more mature retinal whole mount preparations?
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Yes, the JoVE article from Kofuji's lab is a good help, with collagenase / hyaluronidase treatment. We've tested it (with just the collagenase, as we didn't have hyaluronidase in stock), and that helped significantly to go through the ILM.
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I have searched for quite a bit, but have not found much. Does anybody know of an antibody for specifically identifying retinal ganglion cells, which works in birds (or one which works in all amniotes/vertebrates)?
I have only found a few papers in which they used an anti-Brn3A antibody. Does anybody have experience with this?
Thank you!
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It is hard to find such markers unless someone has published the transcriptome of the RGC in birds.
An alternative approach might be to look for RGC specific genes in rodents and check for sequence similarity in birds.
Some examples are: Thy1 and gamma-synuclein.
You might also want to use the Allen Brain Atlas page to look for more such markers in developing or adult mice and then perform ISH or Immuno in RGCs of birds.
Usually, genes involved in specification would be conserved in expression as well.
Hope this helps.
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I would like to test for adhesion and viability of retinal neuronal cells on materials.
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JAM-B cells (also known as J-RGCs) are a molecularly defined subcategory of direction-selective retinal ganglion cells that were first found in mice.
I guess the cells are primary. Not very much sure!!!!