X51 inverted microscope

Serum-free medium precooled at 4 °C was filled to a transwell filter (BD Biosciences, San Jose, CA, USA) to form the chamber. BMSCs (3.0 × 10⁵/mL) were inoculated into the upper transwell chamber, and 10% fetal bovine serum (FBS) was added to the basolateral chamber. The cells were incubated for 24 h at 37 °C in a 5% CO₂ incubator. After culture, the transwell filters were washed twice with sterile PBS and fixed with methanol at room temperature for 30 min. Finally, 0.1% crystal violet was added to stain the cells for 30 min. The cells were observed and photographed under an Olympus X51 inverted microscope (Olympus, Tokyo, Japan).

Immunohistochemical staining of the xenograft tumor was performed on the paraffin-embedded sections. In brief, the sections were dewaxed and rehydrated. For antigen retrieval, the sections were treated with Tris/EDTA at 98°C for 30 min by microwave heating and cooled to room temperature naturally. The sections were incubated in 30% hydrogen peroxide buffer for 20 min to inactive endogenous peroxidase. Then, the sections were blocked using PBS supplemented with 5% normal goat serum at room temperature for 30 min. Then, the sections were incubated with anti-Ki67 (ab16667; Abcam) or anti-PCNA (ab29; Abcam) antibodies diluted in blocking solution at 4°C overnight, followed by incubation with biotinylated secondary antibody for 1 h at room temperature. The sections were then treated with streptavidin-conjugated HRP (CSB-PA644737, CSB-PA489724; CUSABIO, Wuhan, China) for 20 min. The staining intensity was detected using diaminobenzidine (DAB; Dako, Glostrup, Denmark). TUNEL staining was conducted with a DeadEnd Fluorometric TUNEL system (Promega) according to the manufacturer’s protocol. The results were visualized under an X51 inverted microscope (Olympus, Tokyo, Japan).