Immunofluorescence was used to determine the changes in the expression of β-MHC in HL-1 cells in response to different treatments. Briefly, HL-1 cells were grown on cover slips as described previously (18 (link)). All treatments were performed in triplicates. After treatments with Ang II (100nM:12hr) or Neb (1µM: 12hr) coverslips were washed with HEPES (Sigma), fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.5% Triton-X-100, washed with HEPES-T (1mL Tween-20/L), and blocked with 1% bovine serum albumin (BSA) (Jackson ImmunoResearch), 10% goat serum (Sigma) and 0.1% Tween 20 (Fisher Scientific). Cells were incubated with anti- β-MHC antibody (Sigma) (1:100 dilution) overnight at 4°C and repeatedly washed with HEPES. The coverslips were then incubated with Alexa Fluor 488 goat anti-rabbit (Invitrogen Inc.) (1:200 dilution) for 1 hr at room temperature. Coverslips were washed with HEPES and mounted with Fluoroshield with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) and visualized using a Leica DMI 4000B inverted confocal microscope using Leica Application Suite software. Imaging was done at 60x magnification using oil immersion.