Tunel apoptosis detection kit

HE, TUNEL and immunohistochemical (IHC) staining were carried out as reported by us previously [39 (link)]. HE staining was used to detect the pathological changes. Apoptotic cells in tumor tissues were stained with a TUNEL Apoptosis Detection Kit (Beyotime) according to the manufacturer’s protocol. For histological analysis, tumors were fixed overnight in 10% neutral buffered formalin, embedded in paraffin and sectioned at 5-μm thickness using a Leica RM2265 microtome. IHC staining was carried out with an EnVision Detection System HRP. A rabbit/mouse (DAB+) kit (Agilent) was used following the manufacturer’s instructions. Endogenous peroxidase was blocked by incubation with 0.3% hydrogen peroxide for 15 min. Antigen retrieval was performed by boiling the slides in citrate buffer (10 mM, pH 6.0) in a water bath for 20 min. After being rinsed and blocked with 5% bovine serum albumin (BSA), the slides were incubated overnight at 4 °C with primary antibodies, followed by 1 h with labeled Polymer-HRP at room temperature. Subsequently, the slides were exposed to DAB+ Chromogen. Counterstaining with hematoxylin was carried out. After mounting, the slides were observed under an Olympus CX21 microscope, scanned with a high-resolution digital slide scanner (Pannoramic 250, 3DHistech), and quantified by ImageJ software.

A TdT-mediated dUTP Nick End Labeling (TUNEL) Apoptosis Detection Kit (C1086; Beyotime Biotechnology) was used to detect apoptosis in salivary glands. After paraffin sections were dewaxed with xylene, added proteinase K working solution was added to cover the tissue and incubated at 37 °C for 20 min. The sections were washed three times with PBS (pH 7.4). According to the assay kit, TdT and dUTP were mixed together in a ratio of 1: 9 as the TUNEL reaction solution. The tissue sections were covered with TUNEL reaction solution, placed in a humid box, and incubated at 37 °C for 2 h. After washing thrice with PBS, the sections were stained with DAPI solution (C02-04002, Bioss Antibodies, Beijing, China) in the dark for 10 min at room temperature. Finally, a fluorescence quenching agent (S2110, Solarbio, Beijing, China) was used to seal the sections and evaluate the number of TUNEL-positive apoptotic cells using a fluorescence microscope (Olympus, BX51) at × 200 magnification. The number of green fluorescent cells in these areas were considered to be the number of TUNEL-positive cells.

TUNEL staining was performed using a TUNEL apoptosis detection Kit (Beyotime, C1088) according to the manufacturer’s instructions (Ma et al., 2022 ). Images were acquired using a Leica SP5 laser-scanning confocal microscope, and the percentages of positive nucleus were quantified using Image J software.

To analyze the extent of cell death induced by Cortisol, apoptotic cells were stained with the TUNEL method. That is, paraffin tissue sections were stained using the TUNEL apoptosis detection kit (Beyotime Biotechnology, Beijing, China), and nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma, St. Louis, MO, USA) dye.

Human breast cancer cells included MDA-MB-231 cells, MDA-MB-231 luc cells, SUM-159 cells, and SUM-159 luc cells (provided by Xi'an Medical University) were incubated at 37°C with 5% CO₂ in RPMI-1640 (GIBCO) supplemented with 10% fetal bovine serum (FBS, HyClone, Thermo Scientific), penicillin (100 IU/ml), and streptomycin (100 mg/ml). Cells were passaged three times a week. The Britanin working solutions (10 mM Britanin dissolved in DMSO) provided by Shanghai Jiaotong University were prepared by dilution of the stock solution in fresh culture medium on the day of use. Britanin, as a natural product, was purified by high-performance liquid chromatography and characterized by nuclear magnetic resonance (NMR) spectroscopy. The purity of Britanin was greater than 95% (Figure S1). A Cell Counting Kit-8 (Dojindo), Hoechst staining kit (Beyotime), TUNEL Apoptosis Detection kit (Beyotime), D-Luciferin potassium salt (Sciencelight), and antibodies (Abcam) were used in this study. Total RNA RNA extraction and CDNA synthesis used RNAiso Plus and the Reverse Transcription System (TaKaRa, Tokyo, Japan). Quantitative RT-PCR (qRT-PCR) analysis was performed in a 7300 Real-Time System (ABI, New York, America) using the SYBR Green RealMasterMix (TIANGEN, Beijing, China).