Goat anti rabbit dylight 488

Rabbit polyclonal antibody to 14–3-3ζ (cat# ab63635) was purchased from Abcam (Cambridge, MA). Diluted (1:4) whole blood was fixed with formaldehyde then incubated 30 min at RT with rabbit anti-14–3– ζ and CD41-PerCP-Cy5.5 (catalog # 340930, BD Biosciences) in 0.2% Triton X-100, followed by 30 min incubation with goat anti-rabbit Dylight 488 (Abcam, Cambridge, MA). Samples were then diluted with 500 µL HEPES-saline buffer and immediately analyzed by flow cytometry. Control samples containing non-specific rabbit IgG instead of anti-14–3–3 were used to establish levels of non-specific staining.

The expression of A3GALT2 and αGal epitopes in pig cells was analyzed using FACS analysis with A3GALT2 antibody, a synthetic polyclonal antibody against pig A3GALT2 in rabbits, and anti-αGal antibody (Enzo Life Sciences, NY, USA). AECs and PBMCs were stained with mouse anti-αGal epitope antibody for 2 h at room temperature and goat anti-mouse Dylight 488 (Abcam) for 1 h on ice. AECs were stained with A3GALT2 antibody for 2 h at room temperature and goat anti-rabbit Dylight 488 (Abcam) for 1 h on ice. Flow cytometry was performed using BD FACS CantoII (BD biosciences, CA, USA), and the results were analyzed using FlowJo software (version 10; https://www.flowjo.com/).

Coverslips were plated in 6-well plates, and HepG2 cells were added onto them. The following day, cells were fixed with 4% paraformaldehyde, then permeabilized by treating them with PBS containing 0.1% Triton X-100 for 15 min. The slides were washed three times with PBS, then blocked with 2% BSA for 30–45 min at room temperature, The slides were incubated overnight with the following primary antibodies: anti-PRDX2 rabbit monoclonal antibody (1:250, ab109367, Abcam) and anti-β-catenin mouse monoclonal antibody (1:200, ab19381, Abcam) at 4 degrees. The next day, the slides were washed three times with PBST for 5 min each time. Then, the slides were incubated with fluorescent secondary antibody: Goat anti-rabbit-DyLight® 488 (1:1000, ab96899, Abcam) and Goat anti-mouse-DyLight® 647 (1:1000, ab150115, Abcam) for one hour at room temperature in the dark. Afterwards, the slides were washed three times with PBST and observed using a fluorescent microscope.