The problem with antibodies for labeling neurons is that the best markers are not well expressed in neuronal somata.
So for example, the best excitatory marker is Slc17a6 (VGLUT2). Antibodies to VGLUT2 mark terminals, however. Similarly, for inhibitory neurons, GAD1/2 or VGAT are best, but they too are not expressed in the soma.
So, you have two choices.
Either you do in situ hybridization. See this post for an example of VGAT (pan-inhibitory)
In situ hybridization is a powerful method to interrogate gene expression in tissues. It is known as being a challenging procedure due to the many steps where errors and obstacles arise. We've recently begun to use a commercial system called
RNAscope from ACD Bio that gets around many of the pitfalls of traditional ISH.
I've used it a lot now in the spinal cord and DRG. Below is an image of the gene VGAT (Slc32a1) in the dorsal spinal cord. There is nice expression, and most importantly, it w…
ISH is great but a pain in the butt to do traditionally, or expensive with RNAscope.
Your other option is to use transcription factor antibodies. In particular:
Pax2 = Inhibitory. This one works (1:500).
For excitatory in the dorsal horn, TLX3 is used. Unfortunately, the widely used TLX3 antibody is custom from Carmen Birchmaeir’s lab in Germany. I don’t know about commericial.
I made a panel like this here:
I talked about this in another thread
http://forum.painresearcher.net/t/fosgfp-mice-for-noxious-stimulation-some-results/213 Here are some more images. Overall, this FosGFP line is good for activity-dependent labeling. It’s not perfect. There are some Fos+ (IHC) neurons that don’t get labeled, but overall, it’s useful. Could be good for electrophysiology. The stimulus here was 55ºC heat for 30s, followed by perfusion 2 hours after the stimulus. [fos-slices-panel-01-01]
Here is a recent paper doing the same:
A François, SA Low, EI Sypek, AJ Christensen, C Sotoudeh, KT Beier, C Ramakrishnan, KD Ritola, R Sharif-Naeini, K Deisseroth, SL Delp, RC Malenka, L Luo, AW Hantman and G Scherrer,
Neuron, Feb 22 2017
Pain thresholds are, in part, set as a function of emotional and internal states by descending modulation of nociceptive transmission in the spinal cord. Neurons of the rostral ventromedial medulla (RVM) are thought to critically contribute to this process; however, the neural circuits and synaptic mechanisms by which distinct populations of RVM neurons facilitate or diminish pain remain elusive. Here we used in vivo opto/chemogenetic manipulations and trans-synaptic tracing of genetically identified dorsal horn and RVM neurons to uncover an RVM-spinal cord-primary afferent circuit controlling pain thresholds. Unexpectedly, we found that RVM GABAergic neurons facilitate mechanical pain by inhibiting dorsal horn enkephalinergic/GABAergic interneurons. We further demonstrate that these interneurons gate sensory inputs and control pain through temporally coordinated enkephalin- and GABA-mediated presynaptic inhibition of somatosensory neurons. Our results uncover a descending disynaptic inhibitory circuit that facilitates mechanical pain, is engaged during stress, and could be targeted to establish higher pain thresholds. VIDEO ABSTRACT.