Protocol for Fixation of Cultured Cells

1. Grow cells on sterile glass cover slips or slides overnight in 37^oC/5% CO₂ incubator
2. Rinse briefly with PBS

   Option # 1

   3. Fix cells by incubation with 1% formalin in PBS for 10 minutes
   4. Keep wet

   Option # 2

3. Fix cells by incubation with methanol (-10^oC) for 5 minutes
4. Allow to air dry

Option # 3

3. Fix cells by incubation with acetone (-20^oC)
4. Allow to air dry

5. Rinse three times with PBS
6. Incubate for 10 minutes with 0.1% H₂O₂ in PBS to quench endogenous peroxidase activity
7. Rinse once with PBS