Male TIMP-1−/− knockout (KO) mice in the C57BL/6 background (B6.129S4-Timp1tm1Pd/J) and respective TIMP-1+/+ wildtype (WT) C57BL-6 controls were obtained from the Jackson Laboratory. Hepatic IRI was performed as described.4 Briefly, arterial and portal venous blood supplies were interrupted to the cephalad lobes of the liver for 90 minutes using an atraumatic clip and mice Palbociclib in vitro were sacrificed after reperfusion. The animal studies were performed according to approved guidelines by the American Association of Laboratory Animal Care. Serum

alanine transaminase (ALT) and serum aspartate transaminase (AST) levels were measured with an autoanalyzer by ANTECH Diagnostics (Los Angeles, CA), as described.4 Liver specimens were fixed with a 10% buffered formalin solution, embedded in paraffin, and processed for hematoxylin and eosin (H&E) staining; to determine the percentage of necrotic area, 10 random sections per slide were evaluated in duplicate Ceritinib using National Institutes of Health (NIH) Image-J. Immunostaining was performed in cryostat sections as described.4, 11 Mac-1 (M1/70) and Ly-6G (1A8), from BD Biosciences, TIMP-1 (Ab86482; Abcam), MMP-9 (AF909; R&D Systems), and cleaved-caspase-3 (ASP175; Cell Signaling) antibodies were used at optimal dilutions. Sections were

blindly evaluated by counting 10 high-powered fields (HPFs)/section in triplicate. Dual/triple staining was detected by immunofluorescence with Alexa Fluor 594-red antigoat immunoglobulin G (IgG) (H+L) (Molecular Probes), and Texas Red antirat IgG (H+L) antibodies (Vector Laboratories). Alexa Fluor 488 phalloidin (Molecular Probes) and Vectashield mounting media with DAPI (Vector Laboratories) were used for F-actin and nuclear staining, respectively. Slides were analyzed using a Leica Confocal Microscope (UCLA Brain Research Institute). Mice were injected intraperitoneally with 50 mg/kg of 5-bromodeoxyuridine

(BrdU) (Sigma) 2 hours prior to liver harvest as described.12 BrdU incorporation, proliferating cell nuclear antigen (PCNA), and phosphorylated histone H3 were detected by immunohistochemistry MCE公司 in paraffin sections using anti-BrdU (Bu20a; Neomarkers), anti-PCNA (PC-10; Neomarkers), and anti-pH3 (Ser10; Cell Signaling) antibodies. Proliferation indexes were determined in triplicate and quantified under light microscopy by counting 10, randomly chosen, HPFs/section. Data are expressed as the percentage of BrdU, PCNA, or pH3 stained hepatocytes per total number of hepatocytes. MPO activity was evaluated in frozen tissue homogenized in an iced solution of 0.5% hexadecyltrimethyl-ammonium and 50 mmol/L of potassium phosphate buffer solution.4 After centrifugation the supernatants were mixed in a solution of hydrogen peroxide-sodium acetate and tetramethyl benzidine (Sigma).