Primary rabbit monoclonal antibody

Reagents. TQ was obtained from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). Primary rabbit monoclonal antibodies (diluted at 1:1,000) against phosphorylated-LKB1 (3482), LKB1 (3050), phosphorylated-AMPK (9957), AMPK (9957), E-cadherin (3195), Snail (3879), ZEB1 (3396), vimentin (5741) and β-actin (4970) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), the AMPK inhibitor Compound C (ComC) and the AMPK activator AICAR were obtained from Sigma-Aldrich.
Cell culture. HK2, a human renal tubular epithelial cell line and the human RCC cell lines 769-P and 786-O were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). These three cell lines were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 100 µg/ml streptomycin and 100 U/ml penicillin (Invitrogen, Carlsbad, CA, USA). All cells were cultured at 37˚C in a humidified incubator with 5% CO 2 atmosphere.

Formalin-fixed, paraffin-embedded tissue was sliced at 3 μm and stained with hematoxylin-eosin (HE) for observation by light microscopy. Immunohistochemical staining of the tissue sections was performed using the immunoperoxidase method. Briefly, each section was deparaffinized, rehydrated, and incubated with 0.3% hydrogen peroxide in methanol to block endogenous peroxidase activity. Antigen retrieval was performed by autoclaving the sections in 10 mM citrate buffer (pH 6.0) for 5 min at 120°C. All sections were incubated for 10 min with normal horse serum to eliminate nonspecific staining, followed by incubation with a primary rabbit monoclonal antibody (diluted at 1:800; Cell Signaling Technology, Beverly, MA, USA) to detect cleaved caspase 3 (CC3). The antibody was applied overnight to the sections at 4°C, followed by incubation with a secondary antibody (ImmPRESS Reagent Kit; Vector Laboratories, Burlingame, CA) for 60 min. Slides were then incubated in diaminobenzidine (DAB)/Tris solution (3 DAB/Tris tablets diluted in 150 mL distilled water; Muto Pure Chemicals, Tokyo, Japan) supplemented with 15 μL of 30% H 2 O 2 . Finally, the slides were counterstained with hematoxylin. CC3 staining was evaluated in the groups with uterine ischemia for 4 and 8 h, based on our previous finding that the uterus of the cynomolgus monkey can tolerate warm ischemia for up to 4 h.