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Immunocytochemical Staining for Sarcomeric
Alpha
Actin
- Cardiomyocytes are cultured in the 60 mm plate
- 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.
- 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.
- Wash cells 3 times with 1X PBS, 10 min each.
- Incubate with HRP-conjugated secondary Ab against mouse at
RT for 1 hour
- Wash cells 3 times with 1X PBS, 10 min each.
- 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.
- Examine the stained cells under the microscope.
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