Anti mouse cd31 pecam 1 pe

The eyes from adult Tet/opsin/VEGF transgenic mice were prepared into 10-μm-thick slices. These sections were fixed with 4% paraformaldehyde at room temperature, and then rinsed in phosphate buffered saline (PBS). After incubation in 0.5% TritonX-100, the sections were incubated in 5% bovine serum albumin, incubated with polyclonal rabbit anti-mouse-integrin α5, integrin β1, NLRP3, or ASC antibody (Santa Cruz Biotechnology), and then incubated in the mixture of FITC anti-rabbit IgG antibody (Cell Signaling Technology) and anti-mouse CD31(PECAM-1)-PE (eBioscience). After 5 min of incubation in 4′, 6-diamidino-2-phenylindole (DAPI), the sections were photographed with a fluorescence microscope. The HRECs were cultured on the cell culture slide (Millipore) and treated with 20 ng/mL VEGF alone, or in combination with 10 μM ATN-161 for 24 h. After washing with PBS, the HRECs were fixed in 4% paraformaldehyde, and then permeabilized in 0.5% Triton X-100. Subsequently, the HRECs were blocked in 5% bovine serum albumin and incubated in anti-NLRP3 or ASC antibody (Santa Cruz), followed by incubation in the FITC anti-rabbit IgG antibody (Invitrogen). After incubation of 5 min in DAPI, the cell samples were examined under a fluorescence microscope and the images were acquired.

ATN-161 (Qiangyao, St. Louis, MO and China). Primary antibody: integrin α5, NLRP3, ASC (Santa Cruz Biotechnology, Santa Cruz, CA, USA); cleaved-IL-1β (Millipore, Billerica, MA, USA); integrin β1, cleaved-caspase-1 (Abcam, Cambridge, MA, USA); β-actin (Cell Signaling Technology, Inc., Danvers, MA). Secondary antibodies (Cell Signaling Technology, Inc., Danvers, MA). Anti-mouse CD31(PECAM-1)-PE (eBioscience Inc., San Diego, CA); goat anti-rabbit immunoglobulin (IgG)-fluorescein isothiocyanate (FITC) (Santa Cruz Biotechnology). All cell culture reagents were bought from Invitrogen (Carlsbad, CA, USA).