Pelling:Protocols/Methanol Fix

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Methanol is a very fast and easy way to simultaneously fix and permeabilize cells if you DO NOT intend to use phalloidin to stain for actin (MeOH destroys the phalloidin binding site but actin can still be stained using antibodies). MeOH produces excellent microtubule morphology. Pour MeOH into a 50mL centrifuge tube and leave in the -20°C freezer overnight (if not already done). Get and ice bucket ready. Cells from incubator, wash with warm PBS 3x. Pour 2-3 mL of -20°C methanol directly onto cells and immediately place on ice for 3min. Pour off MeOH quickly and forcefully (you may notice a small amount of oily film on the surface - this is lipid, careful not to let it stick to the surface of the dish). Wash 3x with ice cold wash buffer. Incubate with wash buffer for 15min on ice. Proceed to antibody staining as usual.