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Anti-sarcomeric – α actinin and ANP Staining
- 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 eg. alpha sarcomeric actin antibody [alpha Sr-1] (ab28052) and ANP diluted in the blocking buffer as above for 1 hour.
- Wash x3 in PBS for 5 min each.
- 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.