Tritc labeled secondary antibody

After myoblasts were treated with differentiation medium (DM) containing HG-DMEM supplemented with 2% horse serum (HS, Sigma, USA) for the indicated time, the differentiated myoblasts were stained for MyoG or MEF2C using the primary polyclonal antibody MyoG (sc-12732, 1:150, Santa Cruz) or MEF2C (5030S, 1:400, CST) and the appropriate TRITC-labeled secondary antibody (Jackson Lab, 1:500, USA). The nuclei were stained with DAPI. C2C12 myoblasts with only 1–2 nuclei within a cellular structure were evaluated with MyoG or MEF2C staining. MyoG + or MEF2C + cells were defined as differentiated cells that did not fuse to form myotubes. Myoblasts with 3 or more nuclei in the structure of a cell were defined as myotubes. The number of double-positive nuclei in a high-power field (HPF, 50 μm) was analyzed after double staining with MyoG/DAPI or MEF2C/DAPI. Two individuals who were blinded to the results evaluated the images using ImageJ (Java) software (National Institutes of Health, USA).

To detect ClC-5 localization, osteoblast and osteosarcoma cells were fixed and labeled with ClC-5 antibody (1:100) overnight at 4°C, followed by incubation with TRITC-labeled secondary antibody (1:200, Jackson Laboratory, ME, USA) for 1 h at room temperature. Fluorescent images were acquired using the Zeiss LSM 710 laser-scanning confocal microscopy (Munich, Germany).