Add secondary ab in the same blocking buffer (for green ab: CY2 or fluoroscein labeled abs, use 1/200 dilution of secondary ab and for CY3 or Rhodamine, use 1/1000 dilution for 45 min (though these will require some optimization)
Wash in PBS x3
Add DAPI solution to coverslips for 15 min.
Wash x2 in PBS.
Add mounting solution and coverslip. Image cells within 2 days. The α-actinin stain is stable for quite some time but the ANP stain tends to bleach out within about 1-2 weeks.
Immunostaining for ERα or ERb
Fix cells as usual in 3.7% paraformaldehyde in PBS.
Rinse cells twice with PBS after fixation.
Permeabilize and block in 2% FBS / 2% BSA in PBS with 0.1% NP40 for 45 min.
Treat cells with antibodies for α-actinin (1/200 dilution) diluted in the blocking buffer as above for 1 hour.
Wash x3 in PBS for 5 min each.
Then treat cells with anti-ERα (or ERb) antibody at optimized dilution overnight at 4°C.
Wash with PBS x3.
Treat with secondary antibodies as above (same dilutions) for 45 min to an hour.
Wash x3 with PBS.
Add DAPI solution for 15 min.
Wash x3 in PBS, then coverslip.
Dr. Richard Pattern Tufts-New England Medical Centre Molecular Cardiology Research Centre Boston, MA