Anti anp

Immunocytochemistry was performed on in vitro cultured rat H9c2 cardiomyocytes. In brief, cardiomyocytes were washed in PBS and fixed with 4% paraformaldehyde solution for 40 min. Cells were permeabilized with 0.025% Triton-X-100 (v/v) solution in PBS for 40 min and blocked in 1% sterile BSA solution for 60 min at RT. After blocking, cells were washed and incubated in 1:100 diluted primary antibody anti-ANP (cat# GTX109255, GeneTex, United States) in PBS at 4°C for overnight. On the next day, cells were washed in PBS and incubated with 1:200 dilution of anti-rabbit Alexa^®Fluor 488 conjugated secondary antibody (cat# A11008, Life Technologies, United States). Cardiomyocyte nuclei were counterstained with 1 μg/ml DAPI in PBS (cat #A1001, AppliChem, United States) and Alexa^®Fluor 594 Phalloidin (cat# A12381, Life Technologies, United States) was used to counterstain the F-actin filaments. Fluorescence images were captured by Olympus IX71 Imaging Systems (Olympus, United States) and quantified with ImageJ software (NIH, United States).

Protein samples extracted from mouse hearts or NMVCs were used for immunoblotting analysis. The concentration of the proteins was determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein (80 mg) were loaded on a 10% SDS-Tris glycine gel electrophoresis and then transferred onto nitrocellulose membranes. After blocking with 5% nonfat milk for 2 hours at room temperature, the membranes were incubated overnight with the corresponding primary antibodies at 4 °C, including anti-CSE (1:1,000, Abnova), anti-ANP (1:1,000, GeneTex), anti-BNP (1:1,000, ABclonal), anti-MHC (1:1,000, SANTA), and anti-TUBULIN (1:1,000, ABclonal). After being washed with PBST three times, each for 10 minutes, the membranes were incubated with secondary antibodies (1:8,000, ABclonal) for 55 minutes at room temperature and washed with PBST again. The results were detected and analyzed using an Odyssey system (LI-COR Biosciences, Lincoln, NE, USA).