Confocal fluorescence images were collected (excitation 488 nm, emission 505-550 nm) at a rate of one image / 2.5 seconds in a horizontal plane through

the hepatocytes to visualize the canalicular spaces. After establishing a baseline level of fluorescence, 1 μM CGamF was perfused through the chamber Selleckchem BAY 73-4506 and images were collected for 10 minutes. CGamF is a bile acid conjugate that relies on basolateral uptake before being transported across the canalicular membrane into the sealed canalicular vacuole of cultured hepatocytes.24, 25 The increase in fluorescence intensity in the canalicular space over time relative to baseline fluorescence served as a measure of Bsep activity. For LPS-induced cholestasis, rats were anesthetized with isoflurane and injected intravenously with 1 mg/kg LPS, or 0.9% saline as control. After 16 hours, animals were anesthetized with pentobarbital sodium selleck kinase inhibitor (50 mg/kg) and their livers were harvested and snap-frozen in liquid nitrogen for further analysis.26 For estrogen-induced cholestasis, 17 alpha-ethinylestradiol (EE) dissolved in 1,2-propanediol (5 mg/mL) was administered to rats subcutaneously (5 mg/kg/day) for 5 consecutive days as

described.26 Control animals received equivalent amounts of vehicle alone. After 5 days animals were anesthetized and livers harvested as above. Hepatocytes on glass coverslips were washed and then fixed in ice-cold methanol for 5 minutes; rat liver was snap-frozen selleckchem under liquid nitrogen, cryosectioned, and then fixed in 4% paraformaldehyde for 10 minutes. Samples were then permeabilized with 1% Triton X-100, blocked in 2% bovine serum albumin, incubated with primary antibodies for 1 hour at room temperature, washed with phosphate-buffered saline (PBS), incubated with fluorophore-conjugated secondary antibodies for 1 hour at room temperature, washed again with PBS, and then mounted. Negative controls were incubated with secondary antibodies alone. Double- and triple-labeled specimens were examined with a Zeiss LSM 510 Confocal Microscope (Thornwood, NY).23 Liver was homogenized in protein extraction reagent from

Thermo (Rockford, IL). Protein was extracted from isolated hepatocytes in the same buffer. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis on a 4%-20% gel. After transferring to PVDF using a semidry system, the membrane was blocked, incubated with primary antibodies overnight at 4°C, washed, incubated with horseradish peroxidase conjugated secondary antibodies for 1 hour, washed, and then revealed by enhanced chemiluminescence.27 Values listed are mean ± standard error of the mean (SEM). Comparisons were made using Student’s t test or analysis of variance (ANOVA). P < 0.05 was considered significant. Like hepatocytes in intact rat liver, hepatocytes in collagen sandwich culture have a well-developed canalicular network to which canalicular membrane proteins appropriately traffic.