Avidin pink and biotin blue

Intestinal tissue was paraffin-embedded and cut into five-micron-thick longitudinal sections followed by H&E staining. For immunohistochemistry, the un-stained slides were deparaffinized and blocked with 3% hydrogen peroxide in methanol. Antigen retrieval was performed using Diva Decloaking solution (Biocare Medical, Concord CA) (120 °C for 2 min using a pressure cooker). Slides were blocked with avidin-pink and biotin-blue (Biocare Medical), treated with anti-DCLK1 antibody (#62257, 1:400; Cell Signaling Technology, Danvers MA) in DaVinci Green (Biocare Medical), incubated overnight at 4 °C, and visualized with biotinylated goat anti-rabbit IgG (Jackson ImmunoResearch Inc. West Grove PA) followed by streptavidin-horseradish peroxidase (Jackson ImmunoResearch Inc. West Grove PA), diaminobenzidine (Sigma-Aldrich), and hematoxylin counterstaining. Organoid whole-mount immunofluorescence staining was performed according to published method^{17 (link)} with minor modification. Briefly, the organoid was fixed with 10% neutral-buffered formalin, quenched with 50 mM NH₄Cl, permeabilized with 0.5% Triton X-100, blocked with 5% BSA, treated with DCLK1 antibody (#62257, 1:100; Cell Signaling Technology, Danvers MA) and finally visualized with Alexa Fluor^® 488 Conjugate anti-rabbit secondary antibody (#4412, 1:250; Cell Signaling Technology, Danvers MA).