Renalase gtx89570

Fresh ischemia-reperfusion hepatic lobes were collected and rinsed in saline to remove remaining blood. Tissues were cut into 6-μm thick sections with a freezing slicing microtome. The sections were then immersed and fixed in 4% paraformaldehyde at room temperature for one-half hour. Afterwards, the sections were incubated with 5% bovine serum albumin (BSA) and immunolabeled with the indicated antibody (renalase GTX89570, GeneTex) at 4°C overnight. After washing, the sections were incubated with Alexa Fluor 594 goat anti-rabbit IgG (R37117, Invitrogen) for 1 h. After washing, 4′,6-diamidino-2-phenylindole (DAPI) was added to stain the cell nuclei on ice. Tissue fluorescence was imaged on a confocal microscope (Alsi, Nikon). For the quantitative expression of renalase, the density of fluorescence was analyzed by the ImageJ 1.44p software.

As described in previous studies [15 (link), 16 (link)], total tissues were lysed using RIPA buffer, and the protein concentration was determined with a BCA protein assay kit (Pierce Company, Rockford, IL, USA). Protein extracts were used for SDS-PAGE (Invitrogen, Carlsbad, CA, USA), and the proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), which was incubated with primary antibodies (renalase GTX89570, GeneTex, diluted 1 : 1000) overnight at 4°C. After incubation with secondary antibodies (goat IgG antibody GTX228416-01, GeneTex, diluted 1 : 5000) for 1 h at room temperature, the membranes were treated with ECL reagents (170-5061, Bio-Rad, Hercules, CA, USA) prior to visualization using a FluorChem E imager (Cell Biosciences, San Jose, CA) according to the manufacturer's instructions. The specific protein expression levels were normalized to β-tubulin on the same nitrocellulose membrane.