IF Protocol for Cardiomyocytes
GUIDELINE
Immunofluorescent labeling is a straight-forward technique for assessing the presence and the subcellular localization of antigens and / or proteins. Several labeling methods are available depending on the biological sample, cell preparation, and antibodies against the target. The following protocol provides a method describing a suggested protocol for sarcomeric actinin, ANP, ER and ERb fluorescent staining of cardiomyocytes.
METHODS
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-actininand 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 fluorescein labeled abs, use 1/200 dilution of secondary ab and for CY3 or Rhodamine, use 1/1000 dilution for 45 min.
- Wash x3 in PBS.
- 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 x3 with PBS.
- 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.
NOTES
- Reconstitute fibronectin in sterile water at 1 mg/ml. Aliquot and store at -20°C.
- If necessary, store the slides with labeled cardiomyocytes at 4°C for up to 1 month, protecting from light and properly sealing to prevent evaporation.
