Alexa 488 fluorescent secondary antibody

Cells were fixed for 1h in 4% paraformaldehyde (PFA) in phosphate buffer (PB) 0.1 M at pH 7.3 and washed three times in PBS, followed by membrane permeabilization with 0.4% Triton X-100 diluted in PBS at 4 °C for 40 min. A 1% albumin in PBS at 4 °C for 40 min was used to block unspecific binding. After several washes with PBS, the cells were incubated overnight with primary antibodies, 1:500 βIII tubulin (TUJ1; ab78078, Abcam, Cambridge, UK)- a neuronal marker, and 1:500 glial fibrillary acidic protein (GFAP; G3893, Sigma Aldrich, St. Louis, MO, USA), a glial astrocytic marker, in a solution containing 5% normal donkey serum (D9663, Sigma Aldrich, St. Louis, MO, USA) and 0.3% Triton X-100 diluted in PB 0.1M at room temperature. After serial washes with PBS, the cells were incubated with 1:1000 Alexa 488 fluorescent secondary antibody (A11008, ThermoFisher Scientific, Waltham, MA, USA) diluted in 0.3% Triton X-100 on PB 0.1 M for 2 h at room temperature. The samples were counterstained with the nuclear marker 4′-6-diamino-2-phenylindole (DAPI). The same protocol was performed for double IF using 1:1000 Alexa 546 secondary antibody (A11018, ThermoFisher Scientific, Waltham, MA, USA). After washing, cells were imaged with Nikon TS100F inverted microscope (Nikon Instruments Inc., Melville, NY, USA).

Immunostaining was performed on differentiation day 5. Briefly, the cells were fixed with paraformaldehyde (4%, 10 min), permeabilized with Triton X-100 (0.3%, 15 min), blocked with BSA (3%, 2 h), incubated with MyHC antibody (1:100 dilution, 4 °C, overnight), and treated with Alexa 488 fluorescent secondary antibody (Thermo Fisher Scientific) for 1.5 h. For the F-actin staining, the cells were plated in 8-well chamber slides (10³ cells/well) for 24 h, fixed, permeabilized, and stained for 40 min with FITC-conjugated phalloidin (P5282, 50 μg/mL, Sigma) in PBS. The nuclei were counterstained by treating the cells with Hoechst 33342 (Invitrogen) for 15 min. Images were captured randomly using a Leica fluorescence microscope (Microsystems, Mannheim, Germany). The measurements were taken from five separate areas for at least three independent experiments. The MyHC-positive areas, myotube widths, and differentiation and fusion indices were determined as previously described [21 (link)].