Tunel apoptosis assay kit

A TUNEL Apoptosis Assay kit (Roche, Basel, Switzerland) was used to detect the apoptotic cells according to manufactures’ instructions. Briefly, paraffin-embedded sections were deparaffinized and hydrated in a graded ethanol series and then digested with trypsin for 40 min at room temperature. The tissue sections were then incubated with TUNEL reaction buffer in a 37 °C humidified atmosphere for 60 min, and then washed with PBS. TUNEL-positive cells and normal cells in each group were counted under a light microscope at 200× magnification (Olympus, Tokyo, Japan).

TPL and SFN (purity ≥ 98%) were obtained from Shanghai Yuanye Biotech Co., Ltd. (Shanghai, China). Coumarin-6 (C6) was supplied by Sigma-Aldrich Co. (St.Louis, MO, USA). Glyceryl monooleate (MO) was kindly donated by Gattefossé Co. (Lyon, France). Acetonitrile, ethanol and other reagents with analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Cell counting kit-8 (CCK-8) was obtained from Beyotime Biotechnology (Shanghai, China). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco Inc. (Grand Island, NY, USA). TUNEL apoptosis assay kit was obtained from Roche Pharmaceutical Co., Ltd. (Basel, Switzerland). All other chemicals used were of analytical grade.
The Huh-7 cell line and RAW 264.7 cell line were purchased from the Cell Bank of Typical Culture Preservation Committee of the Chinese Academy of Sciences (Shanghai, China). Huh-7 cells and RAW 264.7 cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin in a humidified incubator with 5% CO₂ at 37 °C.
Healthy male Balb/c-nu mice (18 ± 2 g) were randomly assigned to different groups. The experiment was approved by the Ethics Committee of Ninth People’s Hospital, affiliated with Shanghai Jiao Tong University School of Medicine before the research.

The liver tissues embedded in paraffin were sectioned for the TUNEL assay with a commercial TUNEL Apoptosis Assay Kit (Roche Applied Science, Indianapolis, IN, USA). Briefly, the tissue section was dewaxed by washing in xylene and rehydrated through a graded series of ethanol and double distilled water, and then was incubated for 20 min at 37 °C with Proteinase K working solution. After that, the slides were incubated with TUNEL reaction mixture for 60 min at 37 °C in a humidified atmosphere in the dark. Converter-POD was then added on the sample and the slide was incubated in a humidified chamber for another 30 min at 37 °C. Afterwards, the slides were incubated with diaminobenzidine (DAB) substrate solution for 5 min at room temperature in the dark. Sample was counterstained by hematoxylin, rinsed by water and differentiated by 1% hydrochloric acid alcohol solution for a few seconds. Finally, the slides were analyzed by light microscope. For each slide, 10 microscopic fields were randomly chosen for statistical analysis.
The apoptotic index was calculated using the following formula.
$\mathrm{Apoptotic} \mathrm{index} \left(100\%\right) = (\mathrm{number} \mathrm{of} \mathrm{TUNEL} \mathrm{positive} \mathrm{cell} \mathrm{nuclei}/\mathrm{number} \mathrm{of} \mathrm{total} \mathrm{cell} \mathrm{nuclei}) \times 100\%$

Using TUNEL Apoptosis Assay kit (11684795910, Roche, Basel, Switzerland) measured apoptosis, according to the manufacturer’s instructions. Then samples were examined under a fluorescence microscope after anti fluorescence quenching.

Shenfu injection was obtained from China Resources Sanjiu (Ya’an) Pharmaceutical Co. (Shenzhen, China). Lipopolysaccharide (LPS) was from Sigma-Aldrich (St. Louis, MO). Antibodies (p44/42 MAPK(Erk1/2)(3A7)mouse mAb, phospho-p44/42 MAPK(Erk1/2)(thr202/Tyr204), p38, p-p38) used for western blotting (WB) were from Cell Signalling Technology (Danvers, MA). Claudin-4 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Enzyme-linked immunosorbent assays (ELISA) kits for cytokines were purchased from Abcam (Cambridge, MA). TUNEL Apoptosis Assay Kit was obtained from Roche (Basel, Switzerland). Click-iT Plus EdU Assay Kit was from Thermo Fisher Scientific (Waltham, MA).