Donkey anti rabbit igg alexa fluor 594

The slides of ARPE-19 cells and smears of Y79 and WERI-RB-1 cells were fixed with fixed fluid (95% alcohol) for 15 min and washed three times. The cells were permeabilized with 1% Triton X-100 for 10 min. After blocking with 5% bovine serum albumin (BSA) for 2 h, primary antibody (GSDME #13075, Proteintech, China) was added and incubated overnight at 4°C. The primary antibody was washed away, a secondary antibody (donkey anti-rabbit IgG-Alexa Fluor 594, Absin, China) was added for 2 h, and then, the secondary antibody was washed away. The cell nuclei were stained with 4,6-diamino-2-phenyl indole (DAPI) (Servicebio, China), and then the cells were filled with antifluorescence quencher, sealed into tablets, and observed microscopically.

Cells seeded on 12-well plates were fixed with 4% paraformaldehyde (30 minutes), permeabilized with 0.5% Triton X-100 for 5 minutes, and then blocked with 1% BSA in 0.1% PBS-Tween 20 for 1 hour. The cells were then incubated overnight at 4°C with primary antibodies against CD31 (1 : 300, Cat# ab9498, Abcam) or vWF (1 : 500, Cat# ab154193, Abcam), followed by incubation with secondary antibody Donkey anti-Mouse IgG-Alexa Fluor 488 (1 : 1000, Cat# abs20014, Absin) for CD31 or Donkey anti-Rabbit IgG-Alexa Fluor 594 (1 : 1000, Cat# abs20021, Absin) for vWF. Nuclear DNA was labelled with Hoechst (4 μg/ml, Cat# BL801A, Biosharp). Images were investigated under an inverted fluorescence microscope (Ix71, Olympus; Tokyo).

For immunofluorescence assays, HepG2 and Bel-7402 cells were seeded in 12-well plates and treated with GA for 48 h. Then, cells were fixed with 4% paraformaldehyde for 15 min, 0.5%Triton X-100 was used for permeabilization, and 10% normal donkey serum (Invitrogen) was added and incubated for 1 h. Cells were incubated with primary antibody against β-catenin (Cat.NO. 8480S, 1:100; Cell Signaling Technology, United States) at 4°C for overnight and then probed with donkey anti-rabbit IgG-Alexa Fluor 594 (Absin, Beijing, China) in dark at 37°C for 1 h. Finally, the cells were washed with PBS and stained with DAPI (Beyotime). The images were captured by using a confocal microscope (Nikon, Tokyo, Japan). For immunohistofluorescence examination, the specimens were fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. Sections (5 µm) were used to analyze Ki-67 and β-catenin expression. After being counterstained with DAPI, the images were captured using a Zeiss Axiophot two microscope.