Anti col1a1

Cells were washed once with 1 × PBS and fixed in 4% paraformaldehyde (Leagene) for 15 min at room temperature. After washing three times with 1 × PBS, cells were permeabilized with 0.5% Triton X-100 in 1 × PBS for 15 min, washed three times with 1 × PBS, and blocked with 10% goat serum for at least 30 min. Then, all primary antibodies were diluted in 1 × PBS and incubated cells overnight at 4°C. Primary antibodies were anti-Nestin (Millipore, 1:100, rat), anti-Sox2 (abcam, 1:100, mouse), anti-Tuj1 (Santa cruz, 1:100, mouse), anti-NeuN (abcam, 1:300, rabbit), anti-GFAP (Santa cruz, 1:100, rabbit), anti-olig2 (Proteintech, 1:1000, rabbit), anti-TH (Santa cruz, 1:100, rabbit), anti-ChAT (abcam, 1:100, rabbit), anti-GABA (sigma, 1:100, mouse), anti-Nav 1.7 (abcam, 1:100, mouse), anti-COL1A1 (Bioss, 1:400, mouse), anti-Collagen IV (abcam, 1:250, rabbit), and anti-Oct4 (Bioss, 1:400, rabbit). Next, cells were washed three times with 1 × PBS and incubated with secondary antibodies FITC (bioss, 1:100) or Cy3 (cwbio, 1:100) for 30 min at room temperature, followed three 5 min-washed with 1 × PBS. The nuclei were stained with DAPI (BestBio, 1:100) for 15 min.

The protein expression of Mkx and other tendon-specific genes were measured with Western blot using the same condition as described previously [26 (link)]. Proteins were extracted using RIPA Lysis Buffer (Shanghai Weiao Biotechnology Co., Ltd., China). BCA protein reagent kit (Beijing Solarbio Science & Technology Co., Ltd., China) was used to measure the concentration. Thirty micrograms proteins were run on SDS-PAGE gels (8%) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membranes were blocked with 10% skim milk at room temperature for 1 h and were incubated with anti-Mkx (1:200, Aviva Systems Biology, San Diego, USA), anti- Col-1a1 (1:400, Bioss, Beijing, China), anti-Collagen type III (Col-3a1, 1:400, Bioss, Beijing, China), anti-Dcn (1:400, Bioss, Beijing, China), and anti-Tnmd (1:400, Bioss, Beijing, China) primary antibodies solution and anti-β-Actin (1:1000, BOSTER Biological Technology Co., Ltd, China) primary antibody solution overnight at 4 °C. After reacting with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody or goat anti-mouse secondary antibody (BOSTER Biological Technology Co., Ltd, China) for 1 h at room temperature, proteins were detected with ECL hypersensitive chemiluminescence kit (Shanghai Weiao Biotechnology Co., Ltd., China) according to the manufacturer’s recommendations.