Biomicroscopy

The femoral head was fixed with 4% formaldehyde (Solarbio), decalcified with 10% EDTA (Solarbio), dehydrated with gradient alcohol (SINOPHARM, Beijing, China), made transparent using xylene (SINOPHARM), embedded in paraffin (SINOPHARM), and sliced with a Leica pathological slicer (RM2016, Leica, Wetzlar, Germany). The slices were dewaxed in xylene and gradient alcohol in turn. The dewaxed sections were stained according to the HE staining kit (Solarbio) and the Masson staining kit (Solarbio) and then examined under a biomicroscopy (Olympus, Tokyo, Japan).

The bone tissue was fixed, decalcified, dehydrated, made transparent, embedded, sliced, and dewaxed (the procedure was the same as that outlined for H&E staining). Antigen retrieval was performed using the bone tissue antigen retrieval solution (BioShun, Shanghai, China). Then, bone tissues were blocked with goat serum (Solarbio). Rabbit anti-BIRC5 (1/100; ab134170; Abcam) and rabbit anti-GFP (1/1000; ab6556; Abcam) were used for primary antibody reactions. Goat anti-rabbit IgG (Alexa Fluor® 488, 1/800, ab150077, Abcam) and goat anti-rabbit IgG (Alexa Fluor® 594, 1/800, ab150080, Abcam) were used for secondary antibody reactions. DAPI restaining of the nucleus was performed, and the sections were sealed with a sealing solution containing an anti-fluorescent quenching agent (Solarbio), after which images were observed and collected under a biomicroscopy (Olympus).

The bone tissue was fixed, decalcified, dehydrated, made transparent, embedded, sliced, and dewaxed (the procedure was the same as that outlined for H&E staining). Then, the sections were stained according to the instructions of the TUNEL detection kit (Vazyme, Nanjing, China) and sealed with an anti-fluorescence quencher (Solarbio). The images were collected by a biomicroscopy (Olympus).