Gill s haematoxylin

All RNAscope assays were performed following supplier guidelines using the RNAscope 2.5 HD Reagent Kit-RED (322350, ACD, Biotechne) and RNAscope 2.5 HD Detection Reagents-RED (322350, ACD, Biotechne) kits. Following dewaxing, sections were incubated in hydrogen peroxide for 10 min at RT and protease plus reagent for 30 min at 40 °C in a HybEZ oven. RNAscope probes were then applied onto sections for 2 h at 40 °C before incubation with AMP1 (30 min at 40 °C), AMP2 (15 min at 40 °C), AMP3 (30 min at 40 °C), AMP4 (15 min at 40 °C), AMP5 (45 min at RT) and AMP6 (15 min at RT). Slides were then incubated with Fast Red for 10 min at RT and counterstained with Gill’s haematoxylin (Sigma Aldrich). For quantification, scans were uploaded into QuPath software as ‘Brightfield other’ images. Individual ducts were highlighted, and annotations were expanded by 50 µm, with the interior removed to isolate measurements to the myoepithelium and periductal region. Channel 1 was used to detect haematoxylin staining while channel 2 was set to detect red staining (MMP13). Cell segmentation was performed using ‘cell detection’ while chromogen detection was performed using ‘positive cell detection’ with thresholds set according to staining intensity. For DCIS, 12 ducts per patient were annotated and the number of MMP13 positive cells per duct were recorded.

Bovine nasal mucosa were collected for histology examination as described above with a 1 cm square dissected out and snap frozen in liquid nitrogen. Cryosections of 8 μm were fixed with acetone, stained with Vector® Red Alkaline Phosphatase Substrate Kit (Vector Labs, Peterborough, UK), in the presence or absence of levamisole inhibitor (Sigma-Chem Co, Poole, UK) counterstained with Gills Haematoxylin (Sigma-Chem Co, Poole, UK) and mounted with Histomount (National Diagnostics, New Jersey, USA). Images were taken with Olympus BX51 and attached DP71 camera (Olympus, Japan), using Cell^D software (Olympus, Japan).