Franny Jongbloed

133 5 ABSENCE OF PROTEIN AND AMINO ACIDS PROTECT AGAINST HEPATIC IRI Immunohistochemistry Frozen liver sections (5 µm) from different time points (baseline, six and 24 hours after reperfusion) were stained with a monoclonal antibody against neutrophils and visualized with an alkaline phosphatase secondary antibody. Per section two observers blinded to the treatment counted the number of neutrophils in 10 microscopic fields at magnifications of 400x. Microarray analysis For microarray analysis, mice were either fed the control diet or put on either a protein-, Leu-free, Trp-free or Met-free diet for three consecutive days, after which the mice were sacrificed. Directly after each intervention, liver samples were taken and snap frozen in liquid nitrogen for further analyses. RNA isolation of the liver samples was performed as described previously 17,18 . RNA quality was tested via the RNA integrity number (RIN, range 0-10). All samples used scored a RIN between 7.7 and 9.5. Hybridization to Affymetrix HT MG-430 PM Array Plates was performed at the Microarray Department of the University of Amsterdam (the Netherlands), according to Affymetrix protocols and as described previously 17,18 . The procedure for all samples was performed on the same batch. Normalization was done using the Robust Multichip Average (RMA) algorithm at the website www.arrayanalysis.org . Data output consisted of 45,141 probes, whereby more probes could present the same Gene ID. Statistical analysis Data were expressed as mean ± standard error of the mean. Differences in groups were analyzed by Mann-Whitney U tests with SPSS (version 21). Differences were considered significant at P< 0.05. Microarray analyses were performed using the free software package R (R foundation), using the Linear Models for Microarray Data (limma) method with correction for multiple testing using the false discovery rate (FDR) according to Benjamini and Hochberg. Fold changes were expressed as the geometric mean per diet group against the corresponding ad libitum fed control group. An FDR <5% with fold change ≥1.5 were used as cut-off values for significance. The enrichment factor (EF), defined as the number of times an observation was higher than expected by chance, was calculated via the formula: EF=nAB/((nA×nB)/nC), where nA is the number of differentially expressed probe sets (DEPS) in experimental group A, nB the number of DEPS in experimental group B, nC the number of total genes in the microarray, and nAB the number of common DEPS between nA and nB. Functional annotation and analyses were performed QIAGEN’s Ingenuity® Pathway Analysis (IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity ). Subsequent pathway and target categories were generated using the QIAGEN’s Ingenuity Target Explorer (QIAGEN’s, https://targetexplorer.ingenuity.com/) . The prediction inhibition or