Antibody against ki67

SCID/nude mice aged 5-weeks were purchased from the Vital River Laboratory Animal Technology (China). Cells subjected to indicated transfection (100 μL, 1 × 10⁶ cells/mouse) were subcutaneously injected in the left fat pad of mice (n = 8). The width and length of tumors and body weight were measured every three days for 5 weeks. The calculation of tumor volume follows the formation: 0.5 × width² × length. At last, the mice were succumbed to death and tumors were isolated, weighted, and captured. Tumors were cut into small pieces, fixed in 4% PFA, imbedded with paraffin and cut into 5-μm-thick sections for further HE staining and IHC. For HE staining, the tumor sections were subjected to 5% eosin staining solution and hematoxylin. For IHC staining of Ki-67, tissues sections were processed with antigen retrieval, blocking with 5% BSA, and incubation with antibody against Ki-67 (1:1000, Abcam) and secondary antibody for 1 hour at room temperature. The visualization of stained cells was achieved by 3,3-diaminobenzidine (DAB, Beyotime). TUNEL assay was performed by using a TUNEL apoptosis detection kit (Thermo) following manufacturer’s protocol.

HIOEC were seeded on coverslips and were rinsed with PBS and fixed with 4% PFA at room temperature for 15 minutes. Next, the cells were permeabilized with 0.25% Triton X‐100 for 5 minutes, washed with PBS twice, and blocked with 2.5% bovine serum albumin in PBS for 1 hour. Antibody against KI67 (Cat No. ab15580; Abcam, MA, USA) was diluted 100-fold with PBS and incubated at 4 °C overnight.

TA muscle tissues from WT or KO mice were isolated and dehydrated in 30% (wt/vol) sucrose solution. Then, the dehydrated TA muscles were frozen in optimal cutting temperature (OCT) compound. Cross sections (10 μm) of frozen TA muscle were used for H&E staining and immunofluorescence staining. For H&E staining, the slides were first stained in haematoxylin for 8 min, washed in running tap water for 5 min and then counterstained in eosin‐phloxine solution for 1 min. The specimens were dehydrated in graded ethanol (70%–100%) and Xylene, and then covered with mounting medium. For immunofluorescence staining of cryo‐sections, immunofluorescence staining was performed as described in the article.⁴⁷ Immunofluorescence staining of Ki67 and Pax7, cells were fixed with 4% paraformaldehyde (PFA) for 15 min at RT. Then cells were permeabilized with 0.5% Triton X‐100 in phosphate buffer solution (PBS) for 20 min, blocked with 3% IgG‐free bovine serum albumin (BSA) and 0.1% Triton X‐100 in PBS for 1 hour (blocking solution) at RT, followed by incubation with antibody against Ki67 (Abcam) or Pax7 (DSHB) at 4°C overnight. After washing with PBS, the samples were incubated with secondary antibodies (Key Resources Table) for 1 h at RT. Immunofluorescence staining of pS6 (CST) was performed according to the manufacturer's instructions. The nuclei were counterstained with DAPI.

The slides of tumor tissue sections were disposed of with deparaffinization and antigen unmasking, and were then incubated with the antibody against Ki-67 (Abcam, United Kingdom) at 4 C overnight. After washing with PBS, the slides were incubated with Polymer Helper and Poly peroxidase-anti-mouse/rabbit IgG (PV-9000, ORIGENE, China), followed by further incubation with diaminobenzidine (DAB). The tissue sections were mounted after being counterstained with hematoxylin. Some sections were stained with H&E for the histological analysis.