The bungarotoxin works well for AChRs. For motor neurons I have tried all or various combinations of 6 antibodies (SV2 and 2H3 from DHSB, Synapsin1 and MAP2 from Millipore, TUJ1 from Covance, S100 from DAKO ). I tried it on fresh tissues as well as frozen tissues and have tried various scenarios of fixation (4% PFA, from 10 - 40 min) , cryoprotection (20-30% sucrose, from 1 hr to 48 hrs), blocking and antibodies concentrations.
For fresh tissues, I extensively clear them of connective tissues before blocking.
It hardly stains 10-20% of all motor neurons and they dont look nice either.
Any suggestion is highly appreciated,
thanks
thanks
Dear Rizwan,
We have done some very nice immunolabelling of neuromuscular junction in the mice. Please check the details in the following publication.
Best wishes, Refik Kanjhan
Article Glycinergic and GABAergic Synaptic Activity Differentially R...
Thanks Rafik for very useful info. You used embryonic diaphragm and and GM muscles which are thin enough for whole mount. I'm doing adult muscles which are too thick and full of connective tissues. I have a feeling that pre-synaptic antibodies are very poor in penetrating collagens and thick tissues. Would you suggest some way to overcome that ? Manually separating connective tissues and dissecting muscle into small strips helped very little.
Best wishes, Rizwan
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