Fixation and permeabilization protocols from DeMAtteis lab

From LipidomicsWiki

Protocols for cell fixation/permeabilization for IF

• Washes: before fixation cells grown on glass coverslips are washed twice with PBS at RT;
• Fixation: then cells are fixed for 10 min at RT in 4 % paraformaldehyde in PBS (or alternatively for 5 min at –20°C in cold metanol);
• Washes: before permeabilization cells are washed three times with PBS at RT;
• Permeabillzation: cells are incubated for 20 min at RT with a solution containing 0.5% BSA (Bovine serum Albumin), 0,05% Saponin, 50 mM NH4Cl in PBS or alternatively (for 4 min at RT with a solution containing 0.1% Triton X-100 in PBS) use 0.01% (w/v) digitonin in PBS for 30 min at RT
• Washes: cells are washed once with PBS before incubation with primary antibodies.