Monoclonal antibody against sarcomeric α actinin

Ang II treatment was used to induce cardiomyocyte hypertrophy. In our experiments, cardiomyocytes were incubated with 100 nmol/l Ang II for 48 h. The serum-free medium containing Ang II was changed every 24 h. Cardiomyocytes were prepared for immunofluorescence staining, real-time PCR, and Western blot assays. For immunofluorescence staining, monoclonal antibody against sarcomeric α-actinin (Sigma, St. Louis, Missouri, U.S.A.) was added at dilutions of 1:200. Nuclear staining was performed with Hoechst (Sigma, St. Louis, Missouri, U.S.A.). Immunofluorescence was examined under a fluorescence microscope (Zeiss, Heidenheim, Baden-Wuerttemberg, Germany). The surface areas of individual cardiomyocytes were measured using Image-Pro Plus software, which was normalized to control group. To avoid human error, at least five independent zones were selected in one slide and the quantitation was performed blinded by two individuals.

Cardiac myocytes were isolated from the hearts of 1- to 3-day-old neonatal rat pups and plated as previously described^{50 (link)}. Using this method, we routinely obtained cardiac myocyte-rich cultures with > 95% of the cells being cardiac myocytes, as assessed by immunocytochemical staining with a monoclonal antibody against sarcomeric α-actinin (Sigma-Aldrich). Serum-starved cardiac myocytes with 0.1% BSA were incubated for 24 h in the indicated concentrations of βOHB (0–5 mM) and recombinant FGF21 (10 ng/mL, 100 ng/mL).