Mayer s haematoxylin

RNAScope experiments were performed as previously described (Russell et al., 2021 (link)). For the expression analysis of early developmental markers, mRNA expression was assessed using the RNAScope singleplex chromogenic kits (Advanced Cell Diagnostics) on NBF fixed paraffin embedded sections processed as described in the previous section. The probes used for this experiment are listed in Supplemetary File 1 g. ImmEdge Hydrophobic Barrier PAP Pen (H-4000, Vector Laboratories) was used to draw a barrier around section while air-drying following the first ethanol washes. Sections were counterstained with Mayer’s Haematoxylin (Vector Laboratories, H-3404), left to dry at 60 °C for 30 min before mounting with VectaMount Permanent Mounting Medium (Vector Laboratories, H-5000).

All mRNA in situ hybridisations were performed using the RNAscope singleplex or duplex chromogenic kits (Advanced Cell Diagnostics) on formalin fixed paraffin embedded sections processed as described in the above section. The protocol followed the manufacturer’s instructions with slight modifications. ImmEdge Hydrophobic Barrier PAP Pen (Vector Laboratories, H-4000) was used to draw a barrier around section while air-drying following the first ethanol washes. Pretreatment followed the standard length of time for pituitaries (12 min), while embryos were boiled for 10 min. For singleplex, the protocol proceeded to follow the instructions exactly. For duplex, Amplification nine was extended to 1 hr and the dilution of the Green Detection reagent was increased to 1:30. For both protocols, sections were counterstained with Mayer’s Haematoxylin (Vector Laboratories, H-3404), left to dry at 60°C for 30 min before mounting with VectaMount Permanent Mounting Medium (Vector Laboratories, H-5000). Slides were scanned using a Nanozoomer-XR Digital Slide Scanner (Hamamatsu) and processed using Nanozoomer Digital Pathology View (Hamamatsu).

Nasal polyp biopsies were fixed in 10% paraformaldehyde for 18 h. After fixation, the tissue block was embedded in paraffin, then cut in a microtome to 0.4 μm sections and affixed on AEPS (amino-propyl-tri-ethoxy-silane)-coated glass slides. Then, the slides were deparaffinised, rehydrated by washing with xylene and 50% to 100% ethanol concentrations, and finally rinsed with running cold tap water. Heat-based antigen retrieval was done with tris EDTA buffer. Blocking non-specific binding was achieved by incubating the slides with 10% normal serum, and immunohistochemical staining was performed using the Vectastain ABC kit (Vector laboratories) according to manufacturer’s instructions. Sections were incubated with primary antibodies against DMRT3 (PA5-85040; Thermo Fisher, South San Francisco, CA, USA in 1:500 dilution for 40 min. Subsequently, the slides were incubated with biotinylated secondary antibody anti-mouse/rabbit IgG for 30 min to detect DMRT3 in the biopsies. The sections were counterstained using Mayer’s haematoxylin (Vector Laboratories Inc., Burlingame, CA, USA) and DPX-mounted before observation under a microscope.