Values were then normalized to the reference gene to generate gene expression results SAR302503 expressed as a relative ratio. Cleaved caspase 3 and TUNEL Samples of the caudate, right medial, and left lateral liver lobes were paraffin-embedded, serially sectioned at 4 μm, mounted onto positively charge plus slides (VWR) and stained for markers of apoptosis. Deparaffinization and antigen retrieval were performed in 1X Reveal solution using a Decloaking Chamber

(Biocare Medical, Walnut Creek, CA). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide Selleck Natural Product Library (Sigma, St. Louis, MO). The Dako Autostainer (Dako-Cytomation, Carpinteria, CA) was programmed to complete the immunohistochemistry staining for caspase 3. Protein Blocking Veliparib Serum (Dako) was used first to reduce background staining.

Caspase-3 polyclonal antibody (1:200 dilution; Cell Signaling, Beverly, MA) was the primary antibody directed against cleaved caspase-3. The negative control consisted of replacing the primary antibody with non-specific Rabbit IgG antibody (Dako). Biotinylated anti-rabbit immunoglobulin (1:200 diluted in Dako Antibody Diluent) was used as the secondary antibody. Antibody binding was visualized using streptavidin peroxidase (1:200 diluted in antibody diluent) and DAB+ chromogen followed by hematoxylin counterstain. Terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick-end labeling (TUNEL) was performed using the DeadEnd Colorimetric TUNEL system (Promega, Madison, WI). Briefly, sections were rehydrated in decreasing concentration of ethanol followed by a

wash in 0.85% NaCl (Sigma) for 5 minutes. After a final wash in PBS, sections were fixed in 10% formalin in PBS (Richard Allen Scientific, Kalamazoo, MI) for 15 minutes. To help permeabilize tissue, sections were incubated in Proteinase K (Dako) for 20 minutes. The remaining steps including equilibration and end labeling reaction were followed per manufacturer’s protocol (Promega). Apoptotic cells were detected after incubation in DAB chromogen (Invitrogen; Carlsbad, CA) for 2.5 minutes followed with hematoxylin counterstaining (Dako). Clomifene All slides were cover slipped using permanent mounting medium (Richard Allen Scientific). Crude liver ALT quantification Liver tissue (50 mg of each lobe) was weighed and homogenized using the ultra turrax homogenizer in 1 mL buffer (100 mM phosphate buffer at pH 7.4, 0.25 M Sucrose, 0.01 mM EDTA), complete protease inhibitor cocktail tablets (Roche), and 2 mM PMSF. Samples were centrifuged at 2500 g, 4°C for 15 minutes. ALT enzymatic activity in the supernatant was quantified (U/L) using the Hitachi 911 Analyzer (Roche) at 37°C. Pig heart ALT (Roche) of known enzymatic activity was used to verify the performance of the Hitachi 911 in measuring enzymatic activity in crude tissue.