Inos antibody

The compound (E)-N-hydroxy-4-methoxy-2-(biphenyl-4-yl)cinnamide (WK2-16) was provided by Professor Wei-Jan Huang [56 (link)]. Dimethyl sulfoxide (DMSO), LPS and SRB were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Iba1 antibody was obtained from Millipore (Temecula, CA). The GFAP antibody was purchased from ProSci Inc. (Poway, CA, USA). 4′,6′-Diamidino-2-phenylindole (DAPI) was obtained from AAT Bioquest, Inc. (Sunnyvale, CA, USA). HIGHDEF^® IHC Fluoromount was purchased from Enzo Life Sciences (Farmingdale, NY, USA). β-actin, p-p65, p38, and mouse/rabbit IgG antibodies (DyLight 488) were purchased from GeneTex (Irvine, CA, USA). The acetyl-SMC3 antibody was obtained from MBL international (Woburn, MA, USA). The SMC antibody was purchased from Abcam (Cambridge, MA, USA). The COX-2 and p65 antibodies were purchased from Novus Biologicals (Littleton, CO, USA). The iNOS antibody was purchased from Santa Cruz (Dallas, TX, USA). The p-STAT-1, p-STAT-3, p-Akt, Akt, p-ERK, ERK, p-p38 antibodies were purchased from Cell Signaling (Beverly, MA, USA). The horseradish peroxidase (HRP)-conjugated anti-rabbit/mouse secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA).

The proteins in cells of each group were extracted and centrifuged at 4 degrees and then added to the protein lysate for cell lysis with RIPA lysis buffer-combined protease inhibitor cocktail at a ratio of 99 : 1. After that, the total protein was quantified with the BCA method.
The proteins were transferred to the PVDF membrane by electrophoresis with 12% SDS polyacrylamide gel at 300mA2h and then sealed at room temperature with 5% skim milk sealant and TBST for 1 h. The primary antibodies were anti-SIRT1 antibody (diluted 1 : 2000, Abcam, catalog no. AB32441), anti-P65 antibody (diluted 1 : 1500, Abcam, catalog no. AB16502), anti-TNF-α antibody (diluted 1 : 500, Abcam, catalog no. AB6671), anti-IL-6 antibody (diluted 1 : 2000, Cell Signaling Technology, catalog no. 5216), iNOS antibody (diluted 1 : 2000, Santa Cruz Biotechnology, catalog no. SC-650), and GADPH antibody (1 : 5000, Abcam, catalog no. ab9485). All these antibodies were diluted with closed solution, incubated at room temperature for 1 h, and washed with PBS buffer 3 times for 5 min. The polyclonal goat anti-rabbit antibody 1 : 5000 (Cell Signaling Technology) diluted in the closed solution was used as the secondary antibody. Then, it was detected with ECL chemiluminescence reagent imaging (Amersham) and western blotting detection system (Millipore).

Cells were seeded on the glass coverslips in 24-well plates. After washing with PBS three times, cells were fixed with 2% paraformaldehyde for 15 min at room temperature followed by permeabilization with 0.1% Triton × −100. Afterwards, 3% bovine serum albumin (BSA) was used to block the unspecific binding sites for 50 min at room temperature. Cells were then incubated overnight at 4 °C with the primary antibody: 1:100 diluted anti-CD68 antibody (Abcam, MA, USA), 1:50 diluted iNOS antibody (Santa Cruz Biotechnology, CA, USA), 1:50 diluted ARG-1 antibody (Santa Cruz Biotechnology), 1:50 diluted CAMP antibody (Abcam). Samples were then incubated with secondary antibody: 1:200 diluted Donkey Anti-Rabbit IgG H&L, Alexa Fluor^® 594 (Abcam), 1:200 diluted Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594 (Abcam) for 50 min at room temperature. After being rinsed with PBS, 4’, 6-diamidino-2-phenylindole (DAPI) was added to stain the cell nucleus and samples were visualized under immunofluorescence microscopy (Nikon, Eclipse 80i, Tokyo, Japan).