Normal horse serum blocking solution

Cells were plated onto sterile microscopy coverslips and propagated under the experimental conditions (detailed above). Fixation was performed for 10 min in phosphate-buffered saline (PBS) solution containing 4% paraformaldehyde and 10 mM NH4Cl. Cells were permeabilised with 0.2% Triton X-100 in PBS at room temperature for 10 min. Cells were blocked with 2.5% Normal Horse Serum Blocking Solution (Vector Laboratories) for 1 h and stained with a primary antibody directed against TUBB3 in PBS solution containing 1% bovine serum albumin (BSA) and 0.05% Triton X-100. After staining with Cy3-labelled secondary antibody (anti-rabbit) samples were mount onto microscope slides with Vectashield Antifade Mounting Medium containing the 4′,6-diamidino-2-phenylindole (DAPI) stain (Vector Laboratories). Image analysis was performed using ImageJ.

The small intestine was taken and prepared for processing and embedding in paraffin using the Swiss-rolling technique (Bialkowska et al., 2016 (link)), which enables many villi to be investigated in the same cut section. Transverse sections were made using a microtome through the gut rolls at a thickness of 5 µm before mounting on glass slides. Sections were deparaffinized by immersing slides in xylene, then hydrated through 100%, 90%, and 70% ethanol successively. Heat-induced epitope retrieval was performed in citrate buffer (Thermo Fisher Scientific), and then sections were blocked using 2.5% normal horse serum blocking solution (Vector Laboratories) for 1 h at room temperature. Slides were then incubated overnight at 4°C with rabbit anti-mouse Dclk1 (Abcam) or rabbit anti-mouse lysozyme (Abcam) at 1:1,000 in 2.5% normal horse serum blocking solution. Polyclonal rabbit IgGs (Abcam) were used as an isotype control. After washing, sections were incubated with swine anti-rabbit-IgGs/FITC (Agilent Dako), washed, and mounted using Vectashield Vibrance antifade mounting medium with DAPI (Vactor Laboratories). Slides were imaged using a Leica DMi8 inverted microscope and Leica Application Suite (LAS) X software (Leica Microsystems). The resulting image files were analyzed using ImageJ/Fiji.