In situ colorimetric tunel apoptosis assay kit

TUNEL staining was conducted using the in situ colorimetric TUNEL apoptosis assay kit (Beyotime, China), according to the manufacturer's protocol. Briefly, the paraffin sections were deparaffinized and washed. Proteinase K without DNase was added and calibrated at 37°C for 15–30 min. After washing with PBS, we used 3% H₂O₂ (configured with PBS) for incubating the sections at room temperature for 20 min. After inactivated the endogenous peroxidase of the slices, the sections were washed with PBS. Then, the samples were labelled with biotin and stained according to the instructions.

TUNEL staining was performed using the in situ Colorimetric TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer’s instructions. Briefly, the paraffin embedded tissue sections were washed two times with xylene (10 min), dehydrated with ethanol from high to low concentrations, then slices were pretreated with or without DNase proteinase K (20 mg/mL; Millipore, Boston, MA, USA) at 37 °C for 20 minutes. Sections were then washed 3 times with PBS and incubated in 3% hydrogen peroxide solution (3% H₂O₂ in PBS) at room temperature for 20 minutes. Then, 50 L of Streptavidin-HRP working fluid was added to the sample and it was incubated at room temperature for 30 minutes and washed 3 times with PBS. A total of 300 μl of diaminobenzidine (DAB) solution was added, incubating at room temperature for 10 minutes and positive cells were analyzed under light microscopy. The percentage of apoptotic cells was scored as an average of the ratio of TUNEL-positive (TUNEL+) cells to the total number of cells present in each field.

TUNEL staining was performed using the in situ Colorimetric TUNEL Apoptosis Assay Kit (Beyotime), according to the manufacturer's manual. Briefly, incubated the frozen sections in the cold 0.1% Triton X-100 for 2 min. Then moved the slides into the 0.3% H₂O₂ in methanol for 2 min at RT. Washed 3 times with PBS. That was followed by incubation with TdT enzyme solution for 60 min at 37°C. The reaction was terminated by incubation in a stop/wash buffer for 10 min at 37°C. Then used the Streptavidin-HRP for coloration. Incubated the sections in the Streptavidin-HRP working solution for 30 min at RT, following with the colour reagent incubation for 30 min.