Affinipure goat anti mouse igg h l

For immunofluorescence, cells were seeded in 24-well plates. After transfection for 48 h, cells were fixed in 4% formaldehyde for 20 min then washed three times with PBS for 5 min. Subsequently, the cells were permeabilized by adding 0.1% Triton X-100 for 5 min and blocked with goat serum for 30 min. After incubation with MyHC (B103; DSHB, Iowa City, IA, USA; 0.5 μg/mL) at 37 °C for 2 h, the Fluorescein (FITC)-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (Bioworld, Minneapolis, MN, USA; 1:200) or FITC (Bioworld, Minneapolis, MN, USA; 1:50) was added and the cells were incubated at room temperature for 1 h. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China; 1:50) for 5 min. Images were obtained with a fluorescence microscope (Leica, Wetzlar, Germany). The area of cells labeled with anti-MyHC was measured by using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the total myotube area was calculated as a percentage of the total image area covered by myotubes.

Chicken primary myoblast cells cultured in 12-well plates were treated with 4% formaldehyde for 20 min after 48 h transfection and then permeabilized by 0.1% Triton X-100. Subsequently, the cells were blocked with goat serum for 30 min and incubation with anti-MyHC (B103; DHSB, United States; 0.5 μg/ml) overnight. After washed with PBS, the cells were treated with Fluorescein (FITC)-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (BS50950; Bioworld, United States; 1:50) for 1 h. The cell nuclei were stained with DAPI (Beyotime, China) for 10 min and then captured with Leica DMi8 fluorescent microscope. ImageJ software (National Institutes of Health) was used to measure the percentage of the total image area covered by myotubes.

Cells seeded in 12-well plates were fixed in 4% formaldehyde for 20 min and washed three times with PBS for 5 min after 48-h transfection. Subsequently, the cells were treated with 0.1% Triton X-100 for 15 min and blocked with goat serum for 30 min and then incubated with MyHC antibody (B103; DHSB, USA; 0.5 μg/mL) overnight at 4 °C. After that Fluorescein (FITC)-conjugated AffiniPure Goat AntiMouse IgG (H + L) (BS50950; Bioworld, Minneapolis, MN, USA; 1:50) was added to the cells and the cells were incubated at room temperature for 1 h. Besides, the cell nuclei were stained with DAPI for 5 min. And lastly, Leica DMi8 fluorescent microscope (Leica, Wetzlar, Germany) was used to obtain the images, and ImageJ software was used to measure the percentage of the total image area covered by the myotubes.