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?
Try anti-brn3 antibody, if you have a flat-mount retinas, i recommend using of flurogold-back labelling technique that give more visualisation of the dendrites and it stain all ganglion cell types...! Some people also used anti-beta 3 tubular as neuronal marker too... best of luck
In our lab they use a gene gun to biolistically deliver to the GCL and dendrites. However you could consider Golgi staining. It may require some variations in staining methods to get homogenous staining of dendrites in several different ganglion cell types. And of course to allow you to visualize individual dendrites from single cells and their axons may require several retina.
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.
Thanks for the help everyone. I was eyeing some of the older papers using MAP but the figures went black when the document was scanned into the PDF so it's good to hear anti-MAP works!
Yes, I noticed Brn3 is indeed somatic. I've since considered using an SMI-32 (unphosphorylated heavy chain neurofilament) antibody which labels the dendrites/somas/axons of alpha ganglion cells (see link below).
http://www.ncbi.nlm.nih.gov/pubmed/24853938
http://www.ncbi.nlm.nih.gov/pubmed/24853938
Steve, what is the end goal? Are you patching RGCs? Because then I would just fill them with neurobiotin. Have you tried Tuj1? SMI32 will only label a subset of RGCs. There are some Thy1 transgenic lines that label quite nicely and you could try some of those, just check which ones in the literature because they vary in terms of how many they label etc.
I've attached a paper that shows MAP-1 and Brn3a labeling in RGCs in eye with and without optic nerve transection (Mojumder et al, 2008. J Physiol. Fig 10 of the manuscript. Deb Mojumder did the hands-on work shown). Its widefield microscopy of frozen retinal, so its not nearly as sharp as what you would expect if you were to do confocal imaging. Our experience is that MAP labeling is quite robust. We've used both MAP-1 and MAP-2 antibodies and both label pretty well and can be used with paraffin embedded sections.
Hey Tiff, small world.... I am trying to see if a synaptic protein I find in the IPL is localized to retinal ganglion cells. At the moment there is no patching involved, just immunohistochemistry on transverse sections. I will look into TUJ1 some more. Is TUJ1 ganglion cell specific though? As for Thy1, we are looking to try a transgenic.
David, thanks for the info on MAP, I'll definitely give this a go.
I would add to the discussion an observation we made using Brn3a. While staining of mouse cryostat sections showed the expected nuclear labeling in the GCL, we found that the same antibody iabeled also processes when we cultured dissociated PN4 retinal cells on a nanowire array. We used also TRPV4, which labeled the cytoplasm and processes in the GCL. Here is the reference/link if anyone is interested:
Neurite outgrowth and synaptophysin expression of postnatal CNS neurons on GaP nanowire arrays in long-term retinal cell culture. Biomaterials. 2013 Jan;34(4):875-87. doi: 10.1016/j.biomaterials.2012.10.042. PMID: 23131535
http://www.ncbi.nlm.nih.gov/pubmed/23131535
HI, I am looking forward to check for ganglian markers form the mouse retina through western blotting. Can I use MAP-1 or MAP-2 antibodies to do so. Please clarify my doubt. Thanks
Hello, Steve
Did you end up with using any rabbit anti-MAP to label RGC? Could you share the cat# and the staining quality of the antibodies please?
Thanks!
Dahong
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