Changes

*PHEM Buffer (25 mM HEPES, 10 mM EGTA, 60 mM PIPES and 2 mM MgCl, pH 6.9 – only if using paraformaldehyde)*Fixative: Neutral buffered formalinor 4% Paraformaldehyde in PBS or ice cold 10% methanol

*0.1% Triton X-100 in PBS. Can use other permeabilization agents if required*Blocking Solution: 1% BSA and 1% ovalbumin in PBS

#Prepare Cells (For COS or the like, plate the day before to reach ~50%; for 3T3-L1 split one large plate into at required density in 12 wells (6 well plate, 2mL per well) at FBS day 1 and recover onto dishes on ethanol sterilized glass coverslips for 4 days in DMEM/PGS/FBS)

#Wash cells twice with 2 mL ice cold PBS then fix for 10 min at RT#Use neutral buffered formalin, 4% paraformaldehyde in PHEM or ice cold 10% methanolwith desired fixative with gentle rocking

#Add 200 uL of 100mM Glycine in PBSfor PBS for 5 min to quench

#Permeabilize for 10 5 min with Triton X-100 (0.1% in PBS)

#Block for 1-2h with 200 uL of BSA/Ovalbumin (1% of each in PBS) blocking solution#Incubate overnight with primary antibody in blocking solutionat 4C. Dilution varies with the antibody, but typically start with 200x#Wash coverslips 3 times 10 5 minutes with PBS (5 min each)with gentle rocking

#Wash coverslips 3 times 10 minutes with PBS (5 min each)with gentle rocking#Add 10 uL vectashield to glass slide and place cells on slide. Fix with nail polish.