• Products  and  Services  
  • Cell Service
  • Gene Service

 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.
   
   
   Back