I often recur to these forums for tips but this time I must ask anew... I have a student in the lab trying to do an antibody staining against an extracellular epitope of calcium channels. The idea is to look only at channels at the membrane so she has tried to do a live staining at 4C then fixate before the secondary or to first fixate the cells in 2% PFA then apply the primary antibody. In both cases there is a strange INTRACELLULAR signal (confocal microscopy), not seen with the secondary alone. The live staining was, however, done in a saline containing calcium, magnesium, glucose, (the same as she uses for electrophysiological recordings). Could this strange staining be caused by the calcium or other component of the solution? I do not want to have her do it again and again for no good reason... anybody had this type of experience? Would using PBS be more advisable or do you think the issue is other than the solution bathing the cells. But then again, why was the same seen in fixed cells? I have heard that strong fixation can damage membranes, possibly allowing for antibody access to the inside, but this was only 2% PFA...
Any thoughts are very welcome.
And a very Happy New Year!!!