Ab31704

Small “Swiss-rolled” intestinal sections (4 µm) were cut mounted and either stained with Alcian blue and periodic acid-Schiff (PAS, mucus) or left unstained for further processing. Staining of RELM-β was adapted from a published method^{66 (link)}. Sections were stained with primary antibodies, anti-RELM-β antibody (Peprotech 500-P215) or control IgG, followed by incubation with secondary antibody, anti-rabbit IgG biotinylated (Jackson Immunoresearch 711-065-152). Vectastain peroxidase kit (Vector Laboratories PK-6100) and 3,3’-diaminobenzidine were used to reveal the staining and counter-stained with Harris’ solution. The total number of goblet cells in the intestinal epithelial lining was determined by counting the cells immunostained with PAS, and based on their morphology and location being consistent with goblet cells, in one random high-power field (magnification) displaying mucosal integrity and no artefacts. Tuft cells staining was performed using anti-DCLK1 antibody (Abcam, AB31704) and anti-rabbit Alexa Fluor 488 (Jackson ImmunoResearch, 111-545-003). Nuclei were then fluorescently labelled using NucBlue (Invitrogen, Hoechst 33342). The number of tuft cells per villi were counted in five random high-power fields (what magnification) per sample displaying mucosal integrity and no artefacts. These values should be taken as relative differences rather than absolute levels.

Mouse intestines were opened longitudinally and vortexed in a 50-ml conical tube containing Hanks’ balanced salt solution supplemented with 5% heat-inactivated FBS and 10 mM HEPES, pH 7.2. Epithelial cells were isolated by rotating the tissues in a pre-digestion medium (RPMI medium, 5% heat-inactivated FBS, 10 mM HEPES, pH 7.2, and 10 mM EDTA) for 30 min at 37 °. Cells were stained with antibodies for CD326 (BioLegend, catalog no. G8.8, 1:200), CD45.2 (BioLegend, catalog no. 30-F11), anti-DCLK1 (Abcam; ab31704) and LIVE/DEAD.

A pancreatic adenocarcinoma tissue microarray (US Biomax, HPan-Ade 180 Sur-02) containing 180 microsections including 60 paired tumor and normal adjacent tissues was immunostained with anti-DCLK1 antibody (Abcam, ab31704) following our previously described protocol [20 (link)]. Each stained tissue microsection was scored independently by two pathologists and based on percent of tissue demonstrating staining (1 for <10%-4 for > 60%) and staining intensity (1 for lowest intensity, 4 for highest intensity). The resulting scores were multiplied by each other to obtain a composite score.

Tissue was excised and fixed for 48 h in 4% PFA in PBS at 4°C, after which it was transferred to 20% sucrose solution. After cryosectioning, antigen retrieval was accomplished by incubating the slides in 1% SDS for 5 min. Blocking was performed with 10% donkey serum. Following washing, primary antibodies were added and incubated overnight at 4°C. The following primary antibodies were used: rabbit FITC-anti-Lyz (1:400, Dako, F037201), rabbit anti-Muc2 (1:50, Santa Cruz Biotechnology, sc-15334), rabbit anti-ChgA (1:100, Abcam, ab15160) and rabbit anti-Dclk1 antibody (1:1000, Abcam, ab31704). Secondary detection was carried out using Alexa Fluor 488 donkey anti-rabbit secondary antibody (1:500, Thermo Fisher Scientific, A-21206) and DAPI (10 μg/mL). Fluorescent imaging was carried out using a TCS SP5 confocal microscope (Leica). Image analysis was carried out using iMaris software.