Have anyone experienced curved/deformed spindle microtubules after cold methanol fixation (4-5 minutes, -20C)? Anyone knows what is causing this effect on microtubules?
I'm imaging spindle microtubules in a STED microscope and my protocol has worked for a long time in wide field microscopes. Basically, I transfer the coverslips to a well with cold methanol (previously cold at -20C) and fix for 4-5'. After fixation I rapidly transfer the coverslips to another well filled with cytoskeleton buffer. Wash twice with PBS and proceed with the following steps of the IF.
Thanks in advance,
Luísa
So if you image that exact same slide on your widefield scope, it looks OK? If so, then the improved resolution of STED must be revealing sub-optimal structural preservation.
Unless you are stuck with methanol for some other antigen staining, try glut. fixing them instead. I think for ultrastructural work this will work far better than the denaturing induced by methanol.
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