Dnase free protease k

The TUNEL assay was performed to detect in situ apoptosis in the tumor xenograft using a DNA Fragmentation Detection Kit (Beyotime) according to the instructions from the manufacturer. The dewaxed sections were incubated with DNase-free protease K (20 μg/mL, Beyotime) in 10 mM Tris (pH 7.5) at 37 °C for 20 min and washed with PBS. Then, sections were stained with the TUNEL staining mixture at 37 °C for 1 h in the dark, followed by a PBS wash and counterstaining with 4′,6-diamidino-2-phenylindole (DAPI). The stained sections were observed under a fluorescence microscope. Nuclei were stained blue, and apoptotic cells were stained red.

Cell apoptosis in lung tissues was determined by following the instructions of a TUNEL apoptosis detection kit (Millipore, Billerica, MA, USA). Paraffin-embedded lung tissue sections from mice were dewaxed in xylene for 5—10 min, dehydrated with gradient ethanol (90%, 70%), washed with distilled water for 2 min, and reacted with 20 μg/mL DNase-free protease K (ST532, Beyotime Biotechnology, Shanghai, China) at 20—37 °C for 15—30 min. The sections were then incubated in 3% hydrogen peroxide solution prepared with PBS for 20 min at room temperature, biotin-labeled solution for 60 min at 37 °C, and labeling reaction termination solution for 10 min at room temperature. Afterwards, the sections were incubated with 50 μL of streptavidin-horseradish peroxide (HRP) working solution for 30 min at room temperature and developed with 0.2—0.5 mL diaminobezidin (DAB) chromogenic solution for 5—30 min at room temperature. Subsequently, the sections were mounted, observed, and photographed under an inverted microscope, with ten fields of view randomly selected in each group. Positive cells and total cells were counted. Cells with brownish-yellow nuclei were apoptotic-positive cells, while cells with blue nuclei were normal cells. The apoptotic rate was calculated with the following formulae: the number of brown yellow cells/the number of blue cells × 100%.

Brain tissue was embedded in paraffin, then cut into 5 μm slices and baked for 2 h. After brain tissue was dewaxed and hydrated, 20 μg/mL DNase-free protease K (Beyotime, Shanghai, China) was added for 15 min, and 50 μL TUNEL assay solution was added for 60 min, washed with PBS three times. The red signal of the apoptotic cells was observed using a fluorescence microscope.