Icam 1

Western blot analysis was performed using routine protocols. Briefly, protein extracts (50 μg) from the whole eye were separated in 10% SDS-polyacrylamide gels and then transferred onto nitrocellulose membranes. Rabbit polyclonal antibodies against ICAM-1, IFN-γ, IL-10, cleaved-caspase3 (Wanleibio, China), CD11a, CD18, and TNF-α (Affinity Bioscience, USA) were incubated with the membranes overnight at 4°C. The membranes were washed in TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20) 3 times, incubated with the indicated HRP-conjugated goat anti-rabbit IgG (Proteintech) antibody for 1 h at room temperature and then washed in TBST 3 times. A Super Signal Chemiluminescent detection system (Bio-Rad) was used to detect the signals.

The following antibodies were used in the current study: mouse monoclonal antibodies: CD31 (Abcam), Mac-2 (Santacruz, Germany), α-SMA (Boster, Wuhan, China); rabbit polyclonal antibodies: FKBP11 (Bioss, Beijing, China), p65, p-p65 (Cusabio, Wuhan, China), and MMP-9 (Santacruz, Heidelberg, Germany); VCAM-1, ICAM-1, MCP-1 (Wanleibio, Shenyang, China); β-actin (New England Biolabs, Germany); IL1-β (Sigma-Aldrich, Taufkirchen, Germany). The following HRP conjugated secondary antibodies were used for immunoblotting: rabbit IgG (New England Biolabs, Germany), mouse IgG (Abcam). The following secondary antibodies for immunofluorescence were used: Texas red conjugated anti-rabbit and FITC conjugated anti-mouse (Vector Laboratories, CA, USA).
The following reagents used in the current study were: Ang II (Sigma-Aldrich, St. Louis, MO), Trypsin-EDTA and HEPES (Gibco, Germany), fetal bovine serum (Sigma-Aldrich, St. Louis, MO), Protein A-Agarose Immunoprecipitation Reagent (Santacruz, Germany); phosphatase inhibitor cocktail (Roche Applied Science, Penzberg, Germany) and protease inhibitor cocktail (Roche diagnostics GmbH, Mannheim, Germany); BCA reagent (Perbio Science, Bonn, Germany); Vectashield mounting medium with DAPI (Vector Laboratories, CA, USA); PVDF membrane and immobilion enhanced chemiluminescence reagent (Millipore GmbH, Germany).