Rabbit anti crp

To analyze the localization of CRP in the liver tissues, frozen liver sections were treated with the Blocking Solution (DAKO, Glostrup, Produktionsvej, Denmark) at room temperature for 40 minutes, and rabbit anti-CRP (1:50, Santa Cruz) was applied to the sections at 4℃ overnight. Samples were washed with PBS, and incubated with Alexa Fluor 568 (1:100, Invitrogen)-conjugated secondary antibody at room temperature for 1 hour. To detect the co-localization of albumin and CRP in WB-F344 cells incubated with siRNA-CRP and LCA, WB-F344 cells were fixed with 100% methanol (Merck, NJ, USA). The cells were reacted with Blocking Solution (Dako) for 1 hour at room temperature and mouse antialbumin (1:50, Santa Cruz) and rabbit anti-CRP (1:50, Santa Cruz) at 4℃ overnight. After reaction, the cells were incubated with Alexa 488 and 568 (1:100, Invitrogen)-conjugated secondary antibody at room temperature for 1 hour. The slides were stained with 4’, 6-diamidino-2-phenylindole (DAPI). The images were observed with a fluorescence microscope (Nikon, Tokyo, Minato, Japan). All experiments were performed in triplicate.

Cells and liver tissues were lysed in RIPA buffer containing protease inhibitor cocktail (Roche) and phosphatase inhibitor (Sigma-Aldrich). A total of 45 ug protein extracts were separated in 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). The separated proteins were transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). The membrane was incubated with rabbit anti-CRP (1:1,000, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-albumin (1:1,000 Santa Cruz), mouse anti-VEGF, rabbit anti-VEGFR1, mouse anti-endoglin (1:1,000 R&D Systems, Abingdon, UK), and rabbit anti-β-actin (1:3,000, Sigma Aldrich) at 4℃ overnight. The membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (anti-rabbit IgG [1:20,000, Bio-Rad Laboratories, Hercules, CA, USA] or antimouse IgG [1:5,000, Santa Cruz]) for 1 hour at room temperature. The bands were detected using Clarity Western ECL kit (Bio-Rad).