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Recovery and integrity of RNA was determined, and cDNA was made using Omniscript Reverse Transcriptase (Qiagen, Valencia, CA) and 1 ��g of RNA according to the manufacturer��s instructions. Relative gene expression was determined in each cDNA sample using forward and reverse primers designed for each gene of interest (Table?1) and SYBR-Green PCR master mix (Applied Biosciences, CA). Reactions were monitored on an ABI Prism 7000 Sequence Detection System using the cycling parameters: 50��C for 2 minutes, 95��C for 10 minutes, (95��C for 15 seconds, 60��C for 1 minute) for 40 cycles, 60��C for 1 minute, and 60��C dissociation. Relative expression of each gene was normalized to expression of a housekeeping Transferase gene (cyclophilin or 18S ribosomal RNA) to control for intra-/intersample variation and expressed as arbitrary units (AU). Hearts were preserved in 10% neutral buffered formalin for at least 24 hours, processed routinely for histology, cut at 6 ��m, and sections were stained with H&E, Masson��s http://www.selleckchem.com/products/Fasudil-HCl(HA-1077).html trichrome, and von Kossa. Snap-frozen liver samples were preserved for 48 hours in 10% neutral buffered formalin, followed by cryopreservation in 20% sucrose, before routine histology processing and staining with H&E. Tissues were examined by light microscopy in a blinded fashion by?a board-certified anatomical veterinary pathologist (N.D.K.). To determine Kupffer cell infiltration, cells were counted in a single microscopic field for each liver section. Kupffer cell number was normalized to the total number of hepatocytes in each field. To quantitate cardiac fibrosis, trichrome-stained sections were used. An outline of the heart chambers was traced and subtracted from the total heart area. Areas of fibrosis were then traced and normalized to the calculated heart area. Data are expressed as the means �� SEM. Significance of differences was determined for each group of values by analysis of variance (Tukey-Kramer honestly significant difference) or unpaired t-test. A P value learn more performed using GraphPad Prism software version 4 (GraphPad Software, La Jolla, CA). When fed a diet rich in plant sterols, WT mice maintained a normal phenotype, whereas ABCG5/G8 KO mice showed generally poor health, characterized by weight loss, hunching, lethargy, and decreased life span (A.L. McDaniel and H.M. Alger, unpublished observations). Figure?1A shows that WT mice gained weight over the 6-week time-course of?the high-phytosterol diet, whereas the ABCG5/G8 KO mice did not gain weight relative to their baseline measurement. This phenotype was not seen in ABCG5/G8 KO mice fed a diet containing an identical amount of cholesterol (Supplemental Figure?S1 and Supplemental Table S1).