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 (Sigma, Cat#: A2172) 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(Sigma, Cat#: D8001) 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.


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