Liver biochemistry was analyzed by routine automated laboratory a

Liver biochemistry was analyzed by routine automated laboratory assays: total bilirubin, alanine aminotransferase (ALT); aspartate aminotransferase (AST), GGT, and ALP. Blood samples from controls were taken preoperatively. Sepsis was defined according to Bone criteria as suspected or documented

infection on the day of admission to the ICU and fulfillment of at least two of the three criteria for the systemic inflammatory response syndrome (receiving ventilatory support, white-cell count ≤4,000 or ≥12,000 per cubic millimeter, and body temperature ≤36°C or ≥38°C).12 Serum concentrations of cytokines BMS-777607 purchase were quantified by a multiplexed microbead suspension enzyme-linked immunosorbent assay (Biosource, Carlsbad, CA) using the

Luminex 100 system (Austin, TX) as published.13 Individual serum BAs were quantified by high-performance liquid chromatography / mass spectrometry using authentic BA standards and deuterated internal standards.14 Total RNA was isolated and quantified as described.15 Commercial gene expression assays from Applied Biosystems were used and are listed in Supporting Data Table 4. Data are expressed as fold increase relative to the mean of the control patients. Immunoblot analysis of CYP7A1 was performed as described in the online supplement. For histological and immunohistochemical analysis, liver sections from a randomly chosen subset of study patients (40 ICU and 10 controls) were used. Four-μm-thick sections were cut from SAR245409 cost frozen samples and stained with hematoxylin and eosin for a general histological assessment. For evaluation of bilirubinostasis GNAT2 and ductular reaction, sections were stained by Hall’s method and for cytokeratin 7 (CK7) (Dako, Glostrup, Denmark). For immunohistochemistry, 5-μm-thick frozen sections were dried overnight at room temperature, fixed in acetone for 10 minutes, and washed in phosphate-buffered saline (PBS) immediately prior

to use. Sections were incubated with primary antibodies for 30 minutes at room temperature. The primary antibodies used are listed in Supporting Data Table 5. For the staining of CK7, OATP2/8, MRP3, MRP2, MDR1, and MDR3 the second and third step consisted of peroxidase-labeled rabbit anti-mouse and peroxidase-labeled swine anti-rabbit immunoglobulins (both Dako). Secondary and tertiary anti-bodies were diluted (1:50 and 1:100, respectively) in PBS (pH 7.2) containing 10% normal human serum. For the staining of BSEP, the slides were incubated with an anti-rabbit peroxidase-conjugated Envision antibody (Dako) and subsequently incubated with a goat peroxidase anti-peroxidase complex (goat PAP complex; Dako). For NTCP staining a protein block was performed prior to the application of the primary antibody to counteract the strong sinusoidal staining and the secondary step consisted of peroxidase-labeled swine antirabbit IgG (dilution 1:100; Dako), followed by peroxidase-labeled rabbit anti-swine IgG (dilution 1:100; Dako).

Comments are closed.