Trying to find validated antibodies for marking inhibitory and excitatory neuronal populations in the dorsal horn of the spinal cord. Any ideas?
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 was…
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
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.
Here is a recent paper doing the same:
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...
You might also find this useful:
Antibodies are crucial reagents in much of pain research for applications such as immunohistochemistry or western blotting.
Some antibodies are great and others are very poor, and some work only in specific applications.
Here is a spreadsheet that I’ve created, along with some colleagues, that reports on the results of certain antibodies for common pain-related targets. We’ve experimentally validated these. If you’d like to contribute, send me a private message and I’ll add you.