Fast red

MSCs were seeded at 10,000 cells/cm² into a 24-well plate. Medium was changed to osteo-inductive medium (low-glucose DMEM; 10% FBS; 10 nm dexamethasone, Sigma-Aldrich, Gillingham, UK; 50 µg/mL ascorbic acid 2-phosphate, Sigma-Aldrich, Gillingham, UK). Medium was changed every 2 days for a period of 14 days. For qualitative analysis of osteogenic differentiation, cells were fixed in 70% ethanol for 15 min and washed once with ddH2O. After washing, cells were stained with ALP buffer at pH 8.5 (0.2 M Tris, 1 mg/mL fast red, Sigma-Aldrich, Gillingham, UK and 50 µg/mL naphthol phosphate AS-BI, Sigma-Aldrich, Gillingham, UK) for 1 h and imaged under light microscopy.

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.

Immunochemistry was performed on fixed and sectioned B. malayi parasites recovered at various stages of development from intraperitoneally infected jirds, as well as on fixed and sectioned O. volvulus nodules, which were originally obtained from an onchocerciasis patient in Ghana [33 (link),34 (link)]. The parasites were fixed in either 4% formaldehyde or in 80% ethanol then embedded in paraffin. Immunostaining was conducted with the alkaline phosphatase anti-alkaline phosphatase (APAAP) method as previously described [33 (link)]. Primary antibody dilutions of 1:100 to 1:1000 were assessed, and the dilutions 1:200 and 1:500 were found to provide the best signal over background. Polyclonal rabbit anti-mouse IgG 1:25 (Dako, Santa Clara CA, USA) was used as the secondary antibody, then mouse APAAP of at a dilution of 1:40 (Sigma) was applied. The chromatogen Fast Red (Sigma) was used as the substrate and the slides were counter stained with hematoxylin (Merck, Darmstadt Germany). Slides were examined on an Olympus -BX40 microscope and photographed on an Olympus DP70 microscope digital camera (Olympus, Tokyo Japan).

As summarized in Table 1, double-Z oligo probes were designed by the manufacturer (Advanced Cell Diagnostic). The tissue was processed for ISH following the manufacturer's instructions. Briefly, the tissue was fixed tissue in 10% formalin and pretreated with a protease-based solution (pretreatment 4) followed by hybridization at 40°C for 2 h. The probes were mixed using the recommended ratio of 50:1 by volume (c1:c2 probes). Signal amplification was achieved using either diaminodenzidine (chromogenic) or specific fluorophores (FITC and Cy5). Incubation with diaminodenzidine was 10 min. The sections were counterstained with either Fast-Red (Sigma #N3020) or DAPI (Vector Laboratories; H-1500). Totals of 4 and 5 mice were used for the chromogenic and fluorescent ISH, respectively.