Alexa fluor 488 conjugated anti igg

HPCs were incubated in media containing different concentrations of PAI-039 in DMSO for 48 h, fixed with 100% methanol for 5 min at −20°C, and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% bovine serum albumin, and 0.1% Tween for 2 h at room temperature. The HPCs were then incubated with rabbit anti-SERPINE1 (diluted 1 : 100, Abcam, Cambridge, UK) at 4°C overnight. After washing three times in PBS, the primary antibodies were reacted with the corresponding Alexa Fluor 488-conjugated anti-IgG (diluted 1 : 500, Abcam, Cambridge, UK) at 37°C for 30 min. Sections were examined under an Olympus CX41 fluorescence microscope (Olympus, Japan).

The sections were incubated with 5% bovine serum albumin (BSA) for 1 hr at room temperature and then incubated overnight at 4°C with primary antibodies in blocking buffer (Claudin‐5, Santa Cruz Biotechnology; Occludin, Santa Cruz Biotechnology; CD31, Santa Cruz Biotechnology). Then the cords were separately incubated with secondary antibody (Alexa Fluor 488‐conjugated anti‐IgG, Abcam; Texas red‐conjugated anti‐IgG, Santa Cruz Biotechnology). The nuclei were stained with Hoechst 33258 (0.25 mg/ml) dye (Beyotime Institute of Biotechnology, Shanghai, China). For cells, grown on 14 × 14 mm microscopic glass were washed with ice‐cold PBS, fixed with 4% paraformaldehyde for 30 min., then washed with ice‐cold PBS, and blocked in 5% BSA for 1 hr. Then cells were incubated with anti‐p120‐Catennin (Abcam), anti‐beta‐Catenin (Abcam), anti‐Occludin (Santa Cruz Biotechnology), anti‐Claudin‐5 (Santa Cruz Biotechnology) diluted in 1% BSA at 4°C overnight. Cells were washed with PBS followed by incubation with Alexa Fluor 488‐conjugated anti‐IgG or Texas red‐conjugated anti‐IgG secondary antibodies for 1 hr at room temperature. After washing with PBS, the nuclei were stained with Hoechst 33258 (0.25 mg/ml) dye for 7 min., washed with PBS. At last, cells were added with Antifade Mounting Medium (Beyotime Institute of Biotechnology).