Hi everyone.
In the last month I've been trying to do the [35S]GTPgS binding assay on rat brain sections using DAMGO as an agonist, so the goal is to assess the activation of the Mu Opioid Receptors.
The problem is that I've been trying but, no matter what I do, I can never make it work and honestly I don't know what to do anymore.
More precisely the problem that I am repeatedly facing is that I can not see in the sections after the autoradiography any anatomical pattern, so I only see a uniform signal throughout the section, which I guess means that I'm not actually "seeing" the MORs.
I'd like to ask to those of you who know this assay what are the critical steps and aspects I have to pay attention to, the ones that are critical for the assay to actually work.
If you have any tip or suggestion I'm here listening!
Thank you!
Hi Francesco,
I'd be happy to help you troubleshoot. Please provide some specifics on your conditions (buffer; concentration GDP, DAMGO, etc) and the reference for the protocol you're following. How do other brain regions look? For example, there are typical MOR patterns in thalamus and brainstem where receptors are dense and easy to see.
Laura
Dear Francesco, I could try also to help you. Usually in autoradiography the problems arise as a consequence of high basal levels that could be motivated by a plethora of different conditions. Also as Laura indicates the concentration of GDP should be mM and the DAMGO should be 1-100 microM. If you could send an image of your basal and stimulated conditions results we could have a better idea of your problem. The MOR patches in the striatum are a good control indicating the specificity of the results.
Rafael
- Badges
- Science topic