Slcj8103

For ethynyl deoxyuridine (EdU) assay, P7 CKO and WT newborn mice were injected intraperitoneally with EdU (ST067, Beyotime; 20 mg/g) and euthanized 4 h later. As described previously, the spine tissues were fixed, and 6-μm frozen sections were prepared. EdU was detected using the BeyoClick™ Cell Proliferation Kit with Alexa Fluor 555 (C0075S, Beyotime) following the manufacturer's instructions. For the TUNEL assay, we fixed spine tissues of P7 CKO and WT mice and then prepared 6-μm frozen slices. Following the manufacturer's manual, we detected apoptotic chondrocytes in the vertebra using the TdT-mediated dUTP Nick-End Labelling kit (C1088, Beyotime). Briefly, slices were incubated with 20 μg/mL proteinase K for 30 min at 37 °C and then transferred to TUNEL detective solution for 1 h at 37 °C, followed by counterstaining and mounting with DAPI (Sigma, SLCJ8103). Fluorescence microscopy (Olympus, BX63, Japan) was used to capture the immunofluorescence staining images.

Spine tissues of P7 CKO and WT newborn mice were collected, and 6-μm frozen sections were prepared. The sections were permeabilized with 0.1% Triton X-100 for 5 min and blocked in 5% BSA for 30 min at room temperature. Then, sections were incubated with primary antibodies against mouse MAPK7 (Cell Signaling Technology, 3372s; 1:200), COL10A1 (Abcam, ab49945; 1:500), RUNX2 (Abcam, ab192256; 1:400), MMP13 (Abcam, ab39012; 1:400), PTEN (Cell Signaling Technology, 9559s; 1:400), p-AKT (Cell Signaling Technology, 9018s; 1:200), MEF2C (Cell Signaling Technology, 5030s; 1:200), HIF1α (Cell Signaling Technology, 36169s; 1:200), and Cre recombinase (Abcam, ab216262; 1:500) overnight at 4 °C. The following incubation with the secondary antibody Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen, S11227; 1:1000) was performed at room temperature for 1 h. Nuclei were labeled with DAPI (Sigma, SLCJ8103) for 5 min. The immunofluorescence staining images were captured with fluorescence microscopy (Olympus, BX63, Japan) and quantified using Image J software.