Fitc labeled goat anti mouse igg h l

First, the cells in 12-well plate were fixed in 4% paraformaldehyde for 15 minutes and rinsed 3 times for 5 minutes each in PBS. Then, the cells were permeabilized with 0.1% Triton X-100 for 10 minutes, followed by blocking with 1% BSA (Beyotime, Shanghai, China) for 30 minutes. Next, the cells were incubated with anti-MyHC antibody (1:100; Developmental Studies Hybridoma Bank [DSHB], Iowa City, IA, USA) at 4 °C overnight, and then with FITC-labeled Goat Anti-Mouse IgG (H + L; 1:1000; Beyotime) at room temperature for 1 hour. After DAPI (Sigma-Aldrich) staining for 5 minutes, the cell nuclei were examined using a TCS SP8 confocal microscope (Leica, Wetzlar, Germany).

For immunofluorescence, the cells were cultured in 12-well plates, fixed in 4% paraformaldehyde for 15 min, and washed three times (five min each) with PBS. Next, we permeabilized the cells with 0.1% Triton X-100 for 10 min and blocked the cells with 1% albumin bovine serum (Beyotime, Shanghai, China) for 30 min. After incubation with the anti-myosin heavy chain (MHC) antibody (1:100, Developmental Studies Hybridoma Bank, Iowa, USA) overnight at 4 °C, FITC-labeled Goat Anti-Mouse IgG (H + L) (1:1000, Beyotime) was added, and the cells were incubated at room temperature for 1 h. The cell nuclei were stained with DAPI (Sigma-Aldrich, St. Louis, MO USA) for 5 min, and images were obtained with a confocal microscope (TCS SP8, Leica, Wetzlar, Germany).