Isolation and Immunostaining of Mouse Intestinal Organoids

Small intestine crypts were isolated from the small intestine of 4–8-week-old C57BL/6 mice and embedded in a 50% Matrigel (Corning 352231) solution in complete enteroid media consisting of 50 ng/ml recombinant mouse EGF (Peprotech 315-09), 50 ng/ml recombinant murine noggin (Peprotech 250-38), and 50 ng/ml recombinant mouse r-spondin (R&D Systems 7150-RS) as previously described^{80 (link)}. Enteroids were passaged a minimum of one time prior to experimentation in 24-well plates at approximately 150 organoids per well. For whole mount immunofluorescent staining, samples were fixed in 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.1% Triton X-100 for 30 min at room temperature, then blocked in an ice-cold PBS solution containing 5% normal goat serum and 1% BSA for 60 min. Fixed organoids were then incubated overnight at 4 °C with DCLK1 antibody (Abcam, Cat# ab31704, 1:400 dilution) and followed by overnight at 4 °C with Alexa Fluor 488 goat anti-rabbit secondary antibodies (Thermal Fisher, Cat# A11008, 1:2000 dilution). Samples were subsequently counterstained with a 1:5000 DAPI solution (Fisher Scientific 62248) for 1 h at room temperature and stored at 4 °C until visualization using a confocal microscope (Zeiss 710 confocal microscope).