In situ cell death assay kit

Tumor cell apoptosis was detected by in situ cell death assay kit according to the manufacturer’s instructions (Roche, Mannheim, Germany) using TUNEL assay. In brief, the tumor tissue embedded in paraffin was cut into 5 μm-thickness pieces. After that, paraffin section was dewaxed with xylene for 20 min and hydrated with gradient ethanol (100%, 95%, 90%, 80% and 70%). Then, the sections were washed with PBS for three times and permeable with proteinase K at 37°C for 20 min. Thereafter, the sections were fixed with 4% paraformaldehyde for 15 min at room temperature and blocked with 3% H₂O₂ for 10 min. After washing with PBS for three times (5 min per time), the sections were incubated with TUNEL reagent at 37°C for 1 hr. Whereafter, the labeled DNA was observed using a peroxidase-conjugated anti-fluorescein antibody. The percentage of tunel positive cells to the total number of cells is considered to be the apoptotic rate.

According to the manufacturer’s protocol, a commercial kit (in situ cell death assay kit; Roche, Basel, Switzerland) for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was used on paraffin sections of the renal cortical medulla border region. All cells were counted in five different views per section. TUNEL-positive cells were expressed as percentage of total cells.

LPS (from Escherichia coli, 055:B5) and D-Gal were the purchased from Sigma (St. Louis, MO, USA). ELISA kits for detecting the mouse tumor
necrosis factor alpha (TNF-α) and IL-6 were purchased from Neobioscience (Shenzhen, China). Assay kits for measuring alanine aminotransferase (ALT) and
aspartate aminotransferase (AST) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A kit for extracting tissue proteins and assay
kits for determining the activities of caspase 3, caspase 8 and caspase 9 were purchased from Beyotime Biotechnology Institute (Jiangsu, China). An in situ cell
death assay kit for the determination of apoptotic cells was purchased from Roche (Indianapolis, IN, USA). Antibodies against cleaved caspase 3, c-Jun
N-terminal kinase (JNK), phosphorylated JNK (p-JNK), and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). A BCA Protein Assay Kit for
determining the concentration of proteins, horseradish peroxidase-conjugated goat anti-rabbit antibody, and enhanced chemiluminescence (ECL) reagent were
purchased from Pierce Biotechnology (Rockford, IL, USA).

Male BALB/c mice (6–8 wk old, 18-20 g) were provided by the Experimental Animal
Center of Chongqing Medical University. LPS (Escherichia coli,
055:B5), d-GalN and luzindole were purchased from Sigma–Aldrich (St
Louis, MO). The mouse TNF-α and IL-6 ELISA kits were purchased from
NeoBioscience (Shenzhen, PR China). The alanine aminotransferase (ALT) and
aspartate aminotransferase (AST) assay kits were products from the Nanjing
Jiancheng Bioengineering Institute (Nanjing, PR China). The Total Protein
Extract Kit and the Caspase-3, -8, -9 Colorimetric Assay Kit were products from
Beyotime Biotechnology Institute (Jiangsu, PR China). The in
situ cell death assay kit was from Roche (Indianapolis, IN). The
Abs against cleaved caspase-3, poly(ADP-ribose) polymerase (PARP) and β-actin
were obtained from Cell Signaling Technology (Danvers, MA). The HRP goat
anti-rabbit Ab, the BCA Protein Assay Kit and the enhanced chemiluminescence
(ECL) reagent were from Pierce Biotechnology (Rockford, IL).