Which fixative PFA/Methonal/Acetone will preserve the myotube structure of cardiac rat Cells for immunostaining. I've use 4% PFA several times and it detaches about half of the cells, while significantly altering cell morphology. The myotubes, which result from the differentiation of the cardiac cells, are mostly gone after PFA fixation.
I have not used this particular cell type, however I did initially have this same problem with C2C12 mouse myoblasts differentiated into myotubes. The solution was not to change the fixation method, but instead to alter the substrate. I am not sure how you are growing your cells, but I grew mine on sterilized glass coverslips for subsequent imaging. *Freshly* coating coverslips with poly-d-lysine (as in coating them the same day, not purchasing pre-coated or coating and then storing at 4C) and growing them with 10 μg/mL tetrodotoxin to inhibit contraction as myotubes got longer increased the amount of cells remaining on the coverslip after fixation. Additionally, be incredibly gentle when changing solutions on your cells, tilt the culture dish and aspirate liquid from the farthest point away from your coverslip. When adding a new solution pipette it very slowly down the side of the culture dish, never directly down on top of the cells. Good luck!
Dear Jacob, I'm working with satellite cell-derived myotubes from SkMu plated in multiwell plates...A suited strategy that I'm adopting to avoid cell detach is based on the direct addition of PFA 4% without remove media in order to have 2% PFA final concentration.
Reducing PFA concentration can help to preserve your samples
Fix cells for 20' and good luck.
I agree with Alessjo. I will add that the addition of the 4% PFA directly to the media can also reduce surface tension issues that arise from removing the media.
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