Basescope

In situ hybridisation detection for Lgr5 (312178), Olfm4 (311838), Axin2 (400338), Bcl9 (529268) and Bcl9l (466698) mRNA (All Advanced Cell Diagnostics) was performed using RNAscope 2.5 LS (Brown) Detection Kit (Advanced Cell Diagnostics) on a Bond Rx autostainer (Leica) strictly according to the manufacturer’s instructions. Basescope (Advanced Cell Diagnostics) ApcEx14 #701641 (detects wild-type Apc exon 14) was used according to the manufacturer’s instructions.

The BaseScope™ (Advanced Cell Diagnostics, CA, USA) assays were performed manually according to the manufacturer’s instructions. This method allows the detection of exon junctions and the analysis of splice variants. Briefly, the BaseScope™ assay procedure included the following steps: FFPE sections were deparaffinised and treated sequentially with specific pre-treatments to allow for target probe access. Target probes were added onto the slides and incubated in the HybEZ oven (Advanced Cell Diagnostics) for 2 h at 40 °C to allow probe hybridization to RNA targets. The slides were washed and incubated with a series of signal amplification solutions. The signal was amplified using a multi-step process, and detected using a red chromogenic substrate (10 min at room temperature). The slides were counterstained with haematoxylin and mounted with Cytoseal mounting medium (Richard-Allan Scientific, CA, USA).

To measure whether insulin receptors were selectively depleted in sensory tissue, BaseScope (Advanced Cell Diagnostics) mRNA visualization was used. Tissues were fixed in 10% neutral buffered formalin overnight at room temperature. BaseScope assays were performed on paraffin sections (5 μm thickness Superfrost plus slides, Thermo Fisher Scientific) using guidelines provided by the supplier (Advanced Cell Diagnostics). Briefly, slides were baked at 60°C for 1 hour before deparaffinizing in xylene (2 × 5 min) and ethanol (2 × 2 min); they were then dried at 60°C for 5 minutes. RNAScope hydrogen peroxide was applied for 10 minutes at room temperature, then target retrieval was applied for 15 minutes at 100°C. RNAScope protease III was then applied for 30 minutes at 40°C. BaseScope insulin receptor (catalog 719141), positive control (catalog 01071), and negative control probes (catalog 701011) were purchased from Advanced Cell Diagnostics and in situ hybridized to tissue sections with BaseScope Detection Reagent Kit v2 - RED (Advanced Cell Diagnostics). Slides were counterstained with 50% Gill’s hematoxylin and then 0.02% ammonia water before drying for 15 minutes at 60°C and mounting in EcoMount (Biocare Medical).