Microtome sections will be dyed by saturated uranium

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Microtome sections will be dyed by saturated uranium acetate and lead citrate. The sections will be observed by transmission electron microscopy (FEI, Tecnai G2 Spirit Twin). For immunoblot analysis, tissues will be lysed with RIPA buffer (1 mM Tris- HCl, pH=8 (Fisher, Rockford, IL, USA), 1 mM ethylene diamine tetracetic acid 11
(EDTA, Fisher, Rockford, IL, USA), 0.1% sodium dodecyl sulfonate (SDS), 0.1% sodium deoxycholate and 1% Triton-X100 (Sigma, St. Louis, MO, USA)) containing protease inhibitors cocktail (Roche Diagnostics, 05892970001) and phosphatase inhibitor cocktail (Roche Diagnostics, 04906845001). After boiling, tissue lysates will be separated by 8–12% (wt/vol) SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes will be blocked by 5% (wt/vol) non-fat skim milk in TBS-T buffer (50 mM Tris, 150 mM NaCl, 0.05% Tween 20), and then incubated in appropriate primary antibody followed with incubation of secondary antibody diluted in 5% (wt/vol) non-fat skim milk-TBS-T or 5% (wt/vol) BSA-TBS-T. HRP-goat-against-Rabbit/Mouse secondary antibody (#sc-2004/-2005) is purchased from Santa Cruz Biotechnology. For Enzyme-linked immunosorbent assay (ELISA), the levels of IL-1β and IL-18 in liver tissues will be determined by ELISA kits as the manufacturer’s instructions. The optical densities (OD) will be detected at 450 nm by microplate reader. ELISA is determined in triplicate for each sample. The concentrations will be calculated from a standard curve generated from the standards of IL-1β and IL-18. Human IL-1β ELISA Kit (ml710349) and human IL-18 ELISA Kit (ml710351-2) will be purchased from Shanghai Enzyme-linked Biotechnology Co., Shanghai, China. For cytokine/chemokine Luminex assay, peripheral venous blood samples from recipients will be collected using EDTA as an anti-coagulant at 12 hours post graft revascularization. Plasma samples will be thawed completely, mixing well by vortexing and centrifuge prior to use in the assay to remove particulates. Neat plasma samples will be used. Plasma samples will be prepared by centrifugation for 10 minutes at 1000 g and stored at -20°C. Human 38-plex magnetic cytokine/chemokine kits (EMD Millipore, HCYTMAG-60K-PX38) will be used per the manufacturer’s instructions. Complete panel screened for 12
the expression of the following: anti-inflammatory cytokines: IL-1Ra and IL-10; general proinflammatory cytokines: IL-1α, IL-1β, IL-6, IFN-α, TNF/TNF-α, TNF- /lymphotoxin α (LT-α); lymphocyte-associated cytokine: soluble CD40 ligand (sCD40L); Th1/Th17-associated cytokine: IL-12p40; Th1-associated cytokines: IFN-γ and IL-12/IL-12p70; Th2-associated cytokines: IL-4, IL-5, and IL-13; Th9-associated cytokine: IL-9; Th17-associated cytokine: IL-17A; neutrophil-associated chemokines: GRO/CXCL1 and IL-8/CXCL8; eosinophil- associated chemokines: eotaxin/CCL11 and macrophage-derived chemokine (MDC/CCL22); T cell/monocyte–associated chemokines: fractalkine/CX3CL1, IFN-γ–inducible protein-10 (IP10/CXCL10), monocyte chemoattractant protein- 1 (MCP-1/CCL2), and monocyte chemoattractant protein-3 (MCP-3/CCL7); leukocyte-associated chemokines: macrophage inflammatory protein-1 α (MIP-

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