Immunocytochemical Staining for Sarcomeric Alpha Actin

1. Cardiomyocytes are cultured in the 60 mm plate
2. Rinse cells with 1X PBS, fix cells with 3.7% formaldehyde in PBS for 10 min at RT, following 70% ETOH for 5 min, incubate with 0.2% Triton-X 100 in PBS for 5 min, and then wash cells twice with 1X PBS for 5 min.
3.  Incubate cells with 1ml of 2% BSA in PBS for 30 min. Add a primary mouse Ab against sarcomeric alpha actin in the plate for 1 hour at RT, O/N at 4 C.
4. Wash cells 3 times with 1X PBS, 10 min each.
5. Incubate with HRP-conjugated secondary Ab against mouse at RT for 1 hour
6. Wash cells 3 times with 1X PBS, 10 min each.
7. Incubate with 1 ml of DAB solution for 10 min. Wash with 1X PBS.
   * Prepare fresh DAB solution just before you need it:
   * Thaw DAB stock solution at 37 C until it is no longer turbid. Add 5ul of DAB stock solution to 1ml of 1X PBS. Add 1ul of 30% H2O2 and mix well.
8.  Examine the stained cells under the microscope.
    

           BACK