Hrp labelled secondary antibody

Immunohistochemistry was performed as previously described²¹ using rabbit polyclonal antibodies directed against PAX7 (Cell Signaling Technology) and MYH7 (Cell Signaling Technology). We performed immunohistochemical staining on the same paraffin‐embedded tissue blocks that were used for single‐cell sequencing. Before immunostaining, the paraffinized 5 μm sections of skeletal muscle were deparaffinized and submitted to antigen retrieval in Tris‐citrate buffer (Servicebio) followed by incubated with 3% hydrogen peroxide in phosphate‐buffered saline (PBS; Servicebio) to inhibit endogenous peroxidases. After closed with 10% bovine serum albumin (BSA; Servicebio), sections were incubated at 4°C overnight with primary antibodies followed by incubated 1 h with an HRP‐labelled secondary antibody (Cell Signaling Technology) at room temperature. Peroxidase activity was revealed with 3,3′‐diaminobenzidine (DAB; Servicebio), which produces a brown staining, and sections were then stained for 30 s with haematoxylin (Servicebio), dehydrated, and mounted. Finally, the slides were scanned under the E100 microscope (Nikon), and data were analysed using the Aipathwell software (Servicebio).

HUVECs transfected with HIF-2α-overexpressing lentivirus were collected after different hypoxia treatment times. Cells were lysed (60 μL RIPA lysis solution, RIPA:PMSF = 50:1) on ice for 30 min. The lysate was centrifuged at 12,000 rpm and 4 °C for 5 min. Protein was quantified using a BSA assay, and protein samples (50 μg) were loaded and electrophoresed on a 10% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA), which was blocked using 5% milk for 2 h, and incubated with rabbit-anti-HIF-2α, VEGF, NOTCH1, DLL4, Ang2, and β-actin antibodies (all rabbit polyclonal antibodies from KangChen Bio-tech, Shanghai, China) at 4 °C overnight. An HRP-labelled secondary antibody (1:5000; Cell Signaling Technology, Danvers, MA, USA) was added and incubated at room temperature for 1 h. Chemiluminescence, development, and fixation were performed. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to calculate and analyse the OD value of each specific band.