Anti goat igg alexa fluor 555

Established organoids were passaged and replated in a 1:1 ENR:Matrigel solution; 30-μl domes were plated on Nunc Lab-Tek II Chamber Slides and incubated overnight in 200 μl medium at 37°C. Organoids were than treated with 5 ng/ml of IL-13 in ENR or simultaneously with Hpb-CM for 48 h. After stimulation, domes were fixed in 10% formalin for 30 min at room temperature (RT), and permeabilized with PBS-T (PBS + Triton 0.5%) for 15 min. The samples were blocked with 200 μl blocking solution (3% BSA in PBS) for 1 h at RT. Organoids were incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies at RT for 1 h. The nuclei were stained with DAPI (1 µg/ml). Specific antibodies include anti-mouse/rat Ki67 eFluor 660 (Invitrogen), anti-mouse CD326 (EpCAM) Alexa Fluor 488 (BioLegend), goat anti-GFP (Invitrogen), anti-goat IgG Alexa Fluor 555 (Invitrogen), rabbit anti-mouse Dclk (Abcam), rabbit anti-mouse Muc2 (Abcam), and goat anti-rabbit IgG Alex Fluor 555. All fluorescent images were taken on a Zeiss Confocal LSM700, using a 20×/0.8 M27 Plan-Apochromat objective, after mounting with Invitrogen Prolong Glass Antifade Mounting solution. Tile scanning (5 × 5 tiles) was performed as well as Z-stacking to generate images analyzed using Fiji software (Schindelin et al., 2012 (link)).