IF Protocol for Cardiomyocytes

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.

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.

• 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.

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