Mouse anti sox10

Cells fixed in 4% PFA at RT for 30 minutes were permeabilized with 0.1% triton X‐100 in PBS for 5 minutes after washing with 1× PBS. Then, the cells were washed 2 to 3 times and blocked in PBS with 1% BSA at RT for 1 hour. The cells were incubated with primary antibodies: mouse anti‐Sox10 (sc‐365 692, Santa Cruz, 1:200), mouse anti‐MBP (sc‐271 524, Santa Cruz, 1:200) antibodies at 4°C overnight in blocking solution. After washing with 1x PBS, the cells were incubated with Alexa Flour 594‐conjugated secondary antibodies (mouse A11005, Invitrogen, 1:500) or Phalloidin‐iFluor 488 reagent (ab176753, Abcam) at RT for 1 h. For generating imaging data, randomly selected fields (≥3) were photographed using a confocal microscope (Carl Zeiss, LSM 700, Germany) and analyzed using ImageJ software (NIH) in all of imaging data.

Protein extracts were heated to 100 °C for 6 minutes and size fractionated. After blocking with 5% fat-free milk in Tris-buffered saline (1.5 hours, room temperature), extracts were incubated with mouse anti-Sox10 (58 kDa; 1:200 dilution; Santa Cruz Biotechnology) or anti-glyceraldehyde-3-phosphate dehydrogenase (36 kDa; 1:4,000 dilution; ImmunoWay Biotechnology Company, Plano, TX) at 4 °C overnight. Protein bands were detected using a chemiluminescent substrate kit (Pierce, Rockford, IL), and GAPDH served as a control.

Skin cryosections or cultured cells were fixed in 4% paraformaldehyde for 1 h and permeabilized in 0.25% Triton X‐100 for 15 min at room temperature, blocked in 1% BSA for 30 min at room temperature and then stained with Mouse anti‐MyHC (Millipore, 05–716), Rabbit anti‐K14 (Covance, 905301), Mouse anti‐Sox10 (Santa Cruz Biotechnology, SC‐365692), Mouse anti‐Runx3 (R&D Systems, MAB3765‐SP), Rabbit anti‐Mitf (Thermo Fisher, MA5‐32554), Mouse anti‐Pax7 (DSHB), and Rabbit anti‐Laminin (Sigma, L9393) at 4°C overnight with gentle rocking, followed by Alexa 488‐, 561‐ or 647‐labelled anti‐mouse or anti‐rabbit secondary antibody (Invitrogen) staining at room temperature for 1 h. Nuclei were stained with 5 mg/mL DAPI (Invitrogen) for 5 min. All images were acquired by confocal microscopy (Leica). For Pax7 staining, heat‐activated antigen retrieval was performed by placing the paraformaldehyde‐fixed samples in citrate buffer (pH 6.0) at 95°C for 20 min and cooling the slides at room temperature for 20 min, followed by antibody incubation as described above.