Texas red conjugated anti mouse secondary antibody

C2C12 cells were cultured in 8-well chamber slides and immunocytochemistry was performed on day 5. The cells were fixed in 4% paraformaldehyde and incubated with MY32, a primary monoclonal antibody against myosin heavy chain (Sigma-Aldrich, St. Louis, MO, USA), or F12B, anti-myogenin, (Sigma-Aldrich), diluted in PBS/1% BSA/0.3% TritonTM X-100 (Sigma-Aldrich) overnight at 4°C, and then with Texas-red-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch) diluted in PBS/1% BSA/0.3% TritonTM X-100 overnight at 4°C for 1 h. The nuclei were stained with DAPI (Sigma-Aldrich). Images were captured with a BZ-X710 fluorescence microscope, and the fluorescence intensity of the cells and the nuclei count per image with a 20-fold magnification were analyzed using ImageJ (NIH). In addition, the fusion index was defined and determined according to a previous study (24 (link)).

Primary hepatocytes were cultured in 8-well chamber slides and immunocytochemistry was performed on them. Primary hepatocytes were fixed in 4 % paraformaldehyde and incubated with primary monoclonal antibodies: Anti-Cleaved Caspase-3 antibody (ab32042, abcam, Cambridge, UK), diluted in PBS/1%, BSA/0.3%, Triton^TM X-100 (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4°C, and a Texas-red-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch) diluted in PBS/1%, BSA/0.3%, Triton^TM X-100 overnight at 4°C for 1 h. Nuclei were stained with DAPI (Sigma-Aldrich). Images were captured with the BZ-X710 fluorescence microscope, and the ratio of Caspase 3-positive cells per image was analyzed using ImageJ (NIH).