# Anti-GAD67 antibody for inhibitory neuron staining in the dorsal horn? **URL:** \ **Category:** Reagents and Equipment\ **Created:** [December 14, 2016, 11:44pm UTC](https://forum.painresearcher.net/t/anti-gad67-antibody-for-inhibitory-neuron-staining-in-the-dorsal-horn/145 "2016-12-14T23:44:10Z")\ **Posts on this page:** 5\ **Page:** 1
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [December 14, 2016, 11:44pm UTC](https://forum.painresearcher.net/t/anti-gad67-antibody-for-inhibitory-neuron-staining-in-the-dorsal-horn/145/1 "2016-12-14T23:44:10Z")
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. ![](https://canada1.discourse-cdn.com/flex030/uploads/painresearcher/original/1X/8c3257304c119a089d856353c75d3da73e02188e.jpg) 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](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 ![](https://canada1.discourse-cdn.com/flex030/uploads/painresearcher/original/1X/1e37380964b8d50430c0d422a2925db05181f710.png) Does anyone have experience using IHC for inhibitory neurons in the dorsal horn (targeting cell bodies?) ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [December 15, 2016, 1:11am UTC](https://forum.painresearcher.net/t/anti-gad67-antibody-for-inhibitory-neuron-staining-in-the-dorsal-horn/145/2 "2016-12-15T01:11:38Z")
A partial answer to my own question. See this paper: > > > The role of gamma-aminobutyric acid (GABA) in homeostatic control in the brainstem, in particular, in the nucleus tractus solitarius (NTS), is well established. However, to date, there is no detailed description of the distribution of GABAergic neurons within the NTS. The goal of the current study was to reexamine the efficacy of immunohistochemical localization of glutamic acid decarboxylase (GAD) protein, specifically the 67-kDa isoform (GAD67), as a marker for GABAergic neurons in the medulla and to provide a detailed map of GAD67-immunoreactive (-ir) cells within rat NTS by using a recently developed mouse monoclonal antibody. We describe a distribution of GAD67-ir cells in the medulla similar to that reported previously from in situ hybridization study. GAD67-ir cells were localized in regions known to contain high GABA content, including the ventrolateral medulla, raphe nuclei, and area postrema, but were absent from all motor nuclei, although dense terminal labeling was discerned in these regions. In the NTS, GAD67-ir was localized in all subregions. Semiquantitative analysis of the GAD67-ir distribution in the NTS revealed greater numbers of GAD67-ir cells medial to the solitary tract. Finally, dense GAD67 terminal labeling was found in the medial, central, intermediate, commissural, and subpostremal subregions, whereas sparse labeling was observed in the ventral subregion. Our findings support the use of immunohistochemistry for GAD67 as a marker for the localization of GABAergic cells and terminal processes in the rat brainstem. Furthermore, the reported heterogeneous distribution of GAD67-ir in the NTS suggests differential inhibitory modulation of sensory processing. 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. ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [December 20, 2016, 2:54pm UTC](https://forum.painresearcher.net/t/anti-gad67-antibody-for-inhibitory-neuron-staining-in-the-dorsal-horn/145/3 "2016-12-20T14:54:25Z")
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. ---
**Author:** ![alexsjlee](https://avatars.discourse-cdn.com/v4/letter/a/977dab/32.png) [@alexsjlee](https://forum.painresearcher.net/u/alexsjlee)\ **Post date:** [November 29, 2021, 3:20pm UTC](https://forum.painresearcher.net/t/anti-gad67-antibody-for-inhibitory-neuron-staining-in-the-dorsal-horn/145/4 "2021-11-29T15:20:39Z")
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! ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [November 29, 2021, 6:34pm UTC](https://forum.painresearcher.net/t/anti-gad67-antibody-for-inhibitory-neuron-staining-in-the-dorsal-horn/145/5 "2021-11-29T18:34:18Z")
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.