Prolong glass antifade mounting solution

MEFs were plated in a 12-well plate at a density of 15,000 cells per well on top of Poly-D-Lysine/Laminin Cellware 12-mm round coverslips (Corning 354087) and incubated at 37° C overnight. The BMV109 probe was administered the next day and cells were fixed with 4% paraformaldehyde (PFA). MEFs were permeabilized with permeabilization buffer containing .05% TritonX, 1% normal donkey serum, and 1X TBS. They were blocked with a buffer containing 1% normal donkey serum and 1X TBS. Information of antibodies can be found in Table 2. LAMP1 (rat, Developmental Studies Hybridoma Bank 1D4B) and Tubulin- (mouse, Calbiochem cp06) primary antibodies were diluted 1:200 and 1:500 respectively in TBS plus 1% normal donkey serum and 0.1% Triton X-100. Donkey anti-rat AF488 (Invitrogen A48269) and donkey anti-mouse AF568 (Invitrogen A10037) secondary antibodies were diluted 1:2000 in TBS plus 1% normal donkey serum and 1% Triton X-100. MEFs were counterstained with DAPI (Invitrogen D13306) diluted 1:1000 in TBS. Prolong glass antifade mounting solution (Invitrogen P36982) was used prior to coverslipping.

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)).

tamoxifen-inducible Clu fate-mapping mice (Clu-CreERT/+; Rosa26-LSL-tdTomato; Ayyaz et al., 2019 (link)) were infected with 150 Hpb larvae. 2.5 mg tamoxifen (Sigma-Aldrich) was injected i.p. 5, 7, and 12 dpi. To allow the proliferation and differentiation of potential Clu⁺ stem cells, SIs were harvested 12 d after tamoxifen injection for histological analysis. Specifically, the proximal 5 cm of the duodenum was fixed overnight in 10% formalin, transferred to 30% sucrose in PBS for 24 h, frozen in Optimal Cutting Temperature, and kept at −80° until analysis by confocal microscopy. 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 was performed as well as Z-stacking for image analysis using the Fiji software (Schindelin et al., 2012 (link)). Tom⁺ crypts were defined as crypts composed entirely of Tom-labeled cells.