Mouse anti myod

Mouse Anti-Actin, a-Smooth Muscle-Cy3 (1:750, RRID AB 476856; MilliporeSigma, Burlington, MA, USA), rabbit anti-GFP (1:500, RRID AB221569; Thermo Fisher Scientific, Boston, MA, USA), Mouse anti-MYOD (1:500, RRID AB 1098295; Thermo Fisher Scientific, Waltham, MA, USA), Mouse Anti-PITX2 (1:500, RRID AB 509266; Abnova, Taipei City, Taiwan), Alexa-Fluor 546 goat anti-mouse(1:1000, RRID AB 141370; Molecular Probes, Eugene, OR, USA), Alexa-Fluor 647 goat anti-rabbit (1:1000, RRID AB 141663; Invitrogen, Carlsbad, California, USA,), Alexa-Fluor 647 donkey anti-mouse (1:1000, RRID AB 162542; Molecular Probes, Eugene, OR, USA).

Cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. After washing twice with PBS, cells were permeabilized and blocked in PBS containing 0.2% Triton X-100 and 3% donkey serum for 1 h at room temperature. Then, the cells were incubated with primary antibodies at 4°C overnight. After washing three times with PBS, secondary antibodies (Jackson ImmunoResearch) were incubated at 37°C for 1 h. The nuclei were stained with DAPI (Roche Life Science) for 5 min. Primary antibodies were those specific to rabbit anti-SALL4 (Abcam, 1:500), mouse anti-Tubb3 (Biolegend, 1:300), rabbit anti-Synapsin 1 (Abcam, 1:500), rabbit anti-Map2 (Millipore, 1:200), rabbit anti-Neun (Millipore 1:500), rabbit anti-GABA (Sigma, 1:300), anti-mouse neurofilament 200 (Millipore 1:300), rabbit anti-vGlut1 (Synaptic system, 1:300), mouse anti-myosin heavy chain (R&D, 1:300), mouse anti-MyoD (Thermo fisher, 1:200), mouse anti-myogenin (Thermo fisher, 1:200), and mouse anti-α-actinin (Sigma, 1:500). The secondary antibodies used were FITC-conjugated secondary antibodies and TRITC-conjugated secondary antibodies (Jackson ImmunoResearch, 1:200).