Tdt in situ apoptosis detection kit

For immunohistochemistry, paraffin-embedded sections were de-paraffinized in xylene and rehydrated in graded alcohol. Antigen retrieval was done by incubating the sections in citrate buffer pH 6 (Invitrogen) using microwave. Staining was performed using Peroxidase Detection Kit (#1859346-Thermo scientific) with pan-keratin (# PM162AA) antibody (Biocare Medical) according to the manufacturer’s protocol. For the TUNEL assay, TdT In Situ Apoptosis Detection Kit (#4811–30-K) were used according to the manufacturer’s recommendation (R&D System). Briefly, rehydrated lung tissue slides were incubated with Proteinase K solution for 15 min and then washed with deionized water before immersion in quenching solution for 5 min. Following the incubation with 1X TdT labeling buffer for 5 min, tissues were kept in labeling reaction mix for 60 min at 37 ^oC. The reaction was stopped and samples were washed twice with PBS for 5 min. Finally, the slides were incubated with horseradish peroxidase-conjugated streptavidin at 37 °C for 10 min and immersed in Blue label solution for 5 min. After in situ labeling, tissues were counterstained with nuclear fast red, dehydrated, and mounted. The average number of apoptotic cells were calculated by counting three independent area under bright-field microscope.

The BPQD dispersions were purchased from Yuanduo Biotechnology (China). JB was purchased from Desite Biology (China). DMSO and the dialysis membranes (2 kD) were obtained from Solarbio Science & Technology (China). The Annexin V-FITC/PI apoptosis detection kit was purchased from BD Biosciences (China). The ROS test kit was obtained from Beyotime Biotechnology (China). The CCK-8 cell counting kit was purchased from Dojindo Chemical Technology (Japan). Fetal bovine serum (FBS) and RPMI-1,640 were purchased from Biological Industries (Israel). TdT in situ apoptosis detection kit was purchased from R&D Systems (China). DAPI was produced by Servicebio Technology (China). The polycarbonate porous membrane syringe filter (200 nm) was purchased from Whatman (USA).

Geraniin (purity > 99%) was kindly provided by Professor Jikai Liu (Kunming Institute of Botany, Chinese Academy of Science, Kunming, China). Nimodipine injection was obtained from Bayer Pharmaceutical Co., Ltd. (Guangzhou, China). 2,3,5-Triphenyltetrazolium chloride (TTC) was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). SOD and MDA, LDH, NO, and nNOS kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A TdT In Situ Apoptosis Detection Kit was purchased from R&D Systems, Inc. (Minneapolis, USA). An Annexin V-FITC Apoptosis Detection Kit was obtained from KeyGEN Biotech Co., Ltd. (Nanjing, China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were obtained from Gibco (Grand Island, NY, USA). All other chemicals and solvents used were of either analytical grade or pharmaceutical grade.