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==Reagents==
*Fixative: Neutral buffered formalin or 4% Paraformaldehyde (for cytoskeleton bound proteins) in PBS or ice cold 10% methanol(for lipid/membrane bound proteins)
*Cold PBS
*100 mM Glycine in PBS
*0.1% Triton X-100 in PBS. Can use other permeabilization agents if required
*Blocking Solution: 12% BSAPBS
*Vectashield
==Protocol==
#Prepare Cells at required density (quite sparse) in 12 well dishes on ethanol sterilized glass coverslips
#Treat cells as required
#Wash cells twice with 2 mL ice cold PBS then fix for 10 min at RT with desired fixative with gentle rocking
#Wash twice with PBS
#Add 200 uL of 100mM Glycine in PBS for 5 min to quench(up to 1 hour)
#Wash once with PBS
#Permeabilize for exactly 5 min with Triton X-100 (0.1% in PBS)
#Wash three times with PBS (5 min each)
#Block for 1-2h with 200 uL of blocking solution(PBS 2% BSA)#Incubate overnight with primary antibody in 200 ul blocking solution at 4C. Dilution varies with the antibody, but typically start with 200x
#Wash coverslips 3 times 5 minutes with PBS with gentle rocking
#Incubate in 500X secondary solution45 minutes in 2% BSA PBS
#Wash coverslips 3 times 10 minutes with PBS with gentle rocking
#Add 10 uL vectashield to glass slide and place cells on slide. Fix with nail polish, dry for at least 1 hour.
*It is possible to use ImageJ to analyse [[Colocalization]]
[[Category:Immunofluoresence]]