Af594 goat anti mouse

Samples were dewaxed, rehydrated, antigen retrieved and blocked as per immunohistochemical protocol. The primary antibodies were applied overnight at 4 °C (PCK; DAKO, Cat.Z0622, 1:600 dilution and GCTM-5; Millipore Cat.MAB4365, 1:100 dilution). Staining was detected with Alexa Fluor® dyes goat anti rabbit AF488 and goat anti mouse AF594 (Life Technologies, Cat.A11005 and Cat.A11008, 1:400). Note that all antibodies were diluted in DAKO’s antibody diluent (Dako, Cat S2022). Samples were counterstained with DAPI and sealed by a coverslip using an aqueous mounting media (Gelvatol). Positive controls (normal livers with bile ducts) and negative controls for antigen, primary and secondary antibodies were included for all immunofluorescence experiments.

For the immunocytochemical staining, the ZR75-1 cells were cultured in monolayer or suspension for 1, 3, and 7 days. Cells were dissociated with a non-enzymatic dissociation buffer, washed twice, and then re-suspended, at a concentration of 0.5 × 10⁶ cells/mL. Cells were spun onto the microscopy slides using a Thermoscientific Cytospin 4, at 1000 rpm, for three minutes. Cells were then fixed with 4% PFA for 10 min, washed twice, permeabilized with 0.01% Triton-X for 5 min at room temperature, washed twice, and then blocked using 3% BSA, in PBS, for 20 min. Samples were incubated with primary antibodies, overnight, in a blocking solution, at 4 °C. The following day, the slides were washed, twice, with PBS and incubated for an hour with the corresponding conjugated secondary antibodies (goat anti-mouse-AF594 (Life Technologies, A11012) and goat anti-rabbit-AF488 (Life Technologies, A10667). Samples were washed twice and mounted with the DAPI-slow fade mounting solution (Vector Laboratories, H-1200, Burlingame, CA, USA). Confocal imaging was performed using a Zeiss LSM880 (Zeiss, Thornwood, NY, USA). Images were obtained at 40× magnification and analyzed using Zen software (Zeiss). The negative controls were the unlabeled samples and samples stained with only the secondary antibodies.

Secondary antibodies were Alexa Fluor (AF) 488 goat anti-rabbit (CST, 44125), AF 594 goat anti-rabbit (Invitrogen, Thermo Fisher Scientific, A-11012), AF 594 donkey anti-goat (Novus, NBP1-75607), AF 594 goat anti-mouse (Invitrogen, Thermo Fisher Scientific, A-11032), and HRP goat anti-rabbit (Abcam, 205718).