Tunel apopto sis staining kit

Apoptosis is characterized by DNA fragmentation, which can be detected by selective staining. A terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) apopto-sis staining kit (Roche Molecular Biochemicals, Mannheim, Ger-many) was used to detect programmed cell death. The glial cells were treated with hydromorphone (100 nM) for the pre-/per-period with TBH (100 µM) for 30 min), followed by fixation and TUNEL staining. The fixed cells were washed 3 times with PBS containing 0.1 %-Tween 20. The en-zyme and label solutions in the TUNEL stain kit were thoroughly mixed and reacted at 37 °C for 1 h. After the re-action, the cells were washed 3 times with PBS containing 0.1 %-Tween 20 and then examined using a confocal micro-scope. Three slides were examined for each group, and then particles were counted from 10 different parts of each slide.

CD8 immunohistochemistry antibody (cat no.ab4055, Abcam) was used to stain TILs in tumor samples. Brown or yellow‐brown staining of tumor infiltrating cells was defined as positive. CD8 TILs was grouped using a cutoff value of 5% similarly as reported in other studies.¹³, ²⁰ Apoptosis was stained using TUNEL apoptosis staining kit (cat no.11684817910, Roche), and low, medium and high levels were determined as <1%, 1–5% and ≥5%. CD8 and TUNEL immunofluorescence costaining was conducted in 10 randomly selected tumor samples to show the condition of CD8 + T cell apoptosis.