Historically I have always used formaldehyde fixation of cells for immunofluorescence, but lately I have had to use methanol. All of the methods I have found state to do this at -20C, but none of them say why. Just out of curiosity, and so I'm not doing it 'just because', can anyone shed some light on this please?
Thanks!
We also typically used ice-cold MeOH fixation. I think it's a matter of intent. The MeOH denatures proteins, which may or may not be suitable for Ab staining depending upon the epitope. Temp may provide a way to control this. MeOH also permeabilizes and thus extracts components from the cells. Temp (with time) may regulate the extraction efficiency.
We also typically used ice-cold MeOH fixation. I think it's a matter of intent. The MeOH denatures proteins, which may or may not be suitable for Ab staining depending upon the epitope. Temp may provide a way to control this. MeOH also permeabilizes and thus extracts components from the cells. Temp (with time) may regulate the extraction efficiency.
I think the above answer is likely your best bet. Remember that if you cold a chemical reaction it will proceed more slowly. This will also likely apply to kinetics of methanol protein denaturation. Acetone fixation is also done at -20c
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