Methanol fixation works by precipitating proteins, and as such it is a quick method (2-5)minutes is enough time for most antibodies/proteins). Diffuse proteins may be lost,however, in which case PFA fixation may be a better choice. We recommend that you try both types of fixation for your particular antibody/fusion protein when working out your initial immunofluorescence conditions.
MeS Buffer
100 mM MeS, pH 6.9
1 mM EGTA
1 mM MgCl2
Store at 4°C
Methanol Fix (100 ml)
10 ml MeS Buffer
90 ml Methanol
Store at –20°C and use on cells directly from freezer. Pour fix onto cells in dish and leave dish on the bench for 5 minutes, then wash repeatedly with PBS. There is no need for a permeabilization step following this fixation.