Invitrogen evos fl auto

TUNEL staining was used to detect apoptosis in the resected tissue samples. Frozen tumor tissue sections (3–5 µm; stored at −20°C) from the indicated nude mice were processed for TUNEL staining (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Briefly, following fixation by 4% paraformaldehyde for 20–30 min at room temperature, the frozen sections were washed three times with PBS at room temperature (5 min each time). Then, sections were immersed in PBS containing 1% Triton X-100 for 20 min at room temperature. Each sample was incubated with 50 µl TdT enzyme reaction solution (45 µl equilibration buffer, 1 µl biotin-11-dUTP and 4 µl TdT enzyme) for 60 min at RT. Following PBS washing, 50 µl streptavidin-TRITC labeling buffer was added and incubated for 30 min at room temperature. Then, sections were covered with DAPI (1:1,000) for 10 min at RT. Finally, the sections were placed under a cover slip and scanned by Invitrogen EVOS FL AUTO (Thermo Fisher Scientific, Inc.).

Invasion assays were performed using Transwell plates (diameter, 6.5 mm; aperture, 8.0 µm; Corning, Inc.) according to the manufacturer's protocols. Briefly, the filters of the upper chamber were precoated with Matrigel (Becton, Dickinson and Company) at 37°C for 30 min, and 1×10⁴ glioma cells resuspended in 200 µl serum-free medium were added into the chambers. A total of 650 µl culture medium was added to the lower compartments with 10% FBS as a chemoattractant. Following culture at 37°C for 24–48 h, the cells in the upper chambers were removed, and cells that had migrated to the lower membrane were fixed with 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd.) for 20 min and stained with 0.5% crystal violet solution for 30 min at room temperature. The images were scanned by Invitrogen EVOS FL AUTO (Thermo Fisher Scientific, Inc.).