Avidin biotin blocking system

Frozen sections were dried at room temperature and fixed in cold 4% paraformaldehyde (Sigma) in dH₂O at −20° for 5 min. Slides were rehydrated in wash buffer, 0·05% bovine serum albumin (BSA) (Sigma) in PBS, for 5 min. Slides were blocked with 5% BSA in PBS, washed in PBS and avidin/biotin activity was blocked according to manufacturer's instructions with the Avidin/Biotin Blocking System (BioLegend, London, UK). Slides were stained and co‐stained with biotinylated anti‐Siglec F antibody (Bio‐Techne, Abingdon, UK) and fluorescein isothiocyanate‐conjugated cytokeratin antibody (Sigma). Sections were then stained with Streptavidin‐horseradish peroxidase solution (Vector Laboratories, Peterborough, UK), washed with PBS and stained with tyramide‐Cy3 (Perkin Elmer, Seer Green, UK) before being left to air dry. Slides were mounted with VECTASHIELD® HardSet Antifade Mounting Medium with DAPI (Vector Laboratories) and imaged with a Nikon ECLIPSE Ci‐L microscope with a DS‐Fi3 Microscope Camera (Nikon, Kingston upon Thames, UK). Red cells were positive for Siglec F (eosinophils) and green cells were positive for cytokeratin (epithelial cells). Co‐localized staining (i.e. yellow cells) were discarded as tuft cells.

OSCs were fixed with 4% PFA and pre-embedded horizontally in HistoGel™ (Thermo Fisher Scientific, Waltman, MA, USA). The OSCs were then embedded in paraffin with a tissue tamper to ensure horizontal orientation and cut into 5 µm sections. Morphological changes during cultivation were investigated by hematoxylin and eosin (HE)-staining following standard protocols.
For immunohistochemistry, sections were deparaffinized, rehydrated in a descending alcohol series, and treated with 3% H₂O₂ (hydrogen peroxide) for 15 min. Antigen retrieval was accomplished by steaming the sections in a 10 mM citrate buffer (pH 6.0) for 35 min. Unspecific binding was blocked using 10% normal goat serum (Agilent, Santa Clara, CA, USA) in TBS-T (Tris-buffered saline with Tween 20). Sections were then treated with an Avidin/Biotin Blocking System (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions before primary antibodies were applied overnight at 4 °C (Table 1). Afterwards, the sections were incubated with the corresponding biotinylated secondary antibody (Table 1) for one hour, followed by incubation with a VECTASTAIN^® Elite ABC-HRP Kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. Finally, sections were stained with AEC+ High Sensitivity Substrate (Agilent, Santa Clara, CA, USA) and counterstained with hematoxylin.