Anti-GAD67 antibody for inhibitory neuron staining in the dorsal horn?

Experimental Techniques Reagents and Equipment
1

I want to identify inhibitory neurons in the dorsal horn using IHC. I usually see GAD67 or VGAT used as a pan-neuronal marker. I’ve used VGAT ISH (RNAscope) and its excellent and identifies the cell bodies.

dapi-vgat-overlay.jpg1636×1088 449 KB

But I don’t always want to do ISH. I’d like to do IHC if possible. For this, I see people using GAD67.

IHC for GAD67, however, shows mostly GABAergic fibers and projections, but not the soma. I’ve seen this reported elsewhere. Does anyone have any tricks or ways to do this?

I have this anti-GAD67 ab: http://www.emdmillipore.com/US/en/product/Anti-GAD67-Antibody,-clone-1G10.2,MM_NF-MAB5406. It’s been reported in many papers, but mostly in the brain.

It looks great for the fibers and processes. But that’s not exactly what I need. This is with overnight 4C incubation at 1:1000

gad67.png705×469 344 KB

Does anyone have experience using IHC for inhibitory neurons in the dorsal horn (targeting cell bodies?)

2

A partial answer to my own question.

See this paper:

  1. Fresh (not frozen) sections
  2. No Triton-X. Just BSA in PBS for blocking and staining.
  3. 48 hour room temperature incubation!
  4. Monoclonal GAD67 ab from MIllipore.

I already have frozen sections, so not sure what will happen, but I’m re-staining without Triton-X at room temp for 48 hours.

3

Just an update. I tried the 48 hour RT staining. There are indeed more cell bodies, but still many processes and fibers. That might just be a function of the neuroanatomy of the DH, no matter what. I will try one day with non-frozen sections and see what happens.

4

Hi Alex! i am trying to see if GAD67 can work for cell bodies as well!
Would you be able to update me if the non frozen sections worked better when you get a chance?
Thank you!

5

Hi Alex,
I don’t think I ever got it to work. If I wanted to see cell bodies, I used transgenic mice or RNAscope.

1 Like