I am hoping someone here can give me a little help with my NMJ staining.
I am staining mouse so keys and tibialis anterior muscle. The stain works well but even though I have teased the muscles into smaller bundles when I go to coverslip they do not flatten well resulting in air pockets ect. I can still image them acutely but long term they don’t keep as well. I am quite experienced in staining nervous tissue sections and so have never had this problem.
I am worried about teasing the muscle into too small of bundles - is this possible? Or is it better to go smaller and have more individual bundles, thanks very much in advance!
The mouse tibialis anterior (TA) muscle is too big and asymmetric to put under a coverslip. If I was going to try and put a mouse muscle under a coverslip, I would use a soleus (preferably) or extensor digitorum longus muscle. I have imaged NMJ before in TA muscle using fluorescently-labeled tetrodotoxin. I used a confocal microscope with a 10X dipping objective lens. The mouse's leg was placed in a bath containing Krebs-Ringer. The procedure worked well except that only a few NMJ on the surface of the muscle were in the focal plane at a given time.
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