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 (for lipid/membrane bound proteins) in PBS or ice cold 10% methanol (for cytoskeleton bound proteins)

*0.1% Triton X-100 in PBS. Can use other permeabilization agents if required*Blocking Solution: 12% BSA and 1% ovalbumin in PBS

#Prepare Cells at required density (For COS or the like, plate the day before to reach ~50%; for 3T3-L1 split one large plate into quite sparse) 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(up to 1 hour)

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

#Block for 1-2h with 200 uL of BSA/Ovalbumin blocking solution (1PBS 2% of each in PBSBSA) blocking solution#Incubate overnight with primary antibody in 200 ul 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#Incubate in 500X secondary solution45 minutes in 2% BSA PBS#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(coverslip side up, use sharp tweezers and tip, 2 coverslips on each slide). Label slide with date, cells and treatment + color code for antibodies. Fix with nail polish, dry for at least 1 hour up to ON in dark drawer. Store long term in sealed box at 4C.