Endogenous avidin biotin

CD31-positive blood vessels were stained following the manufacturer’s instructions. In brief, sections were deparaffinised and heat-mediated antigen retrieval in 10 mM citrate buffer (pH6) was performed for 10 minutes. Endogenous peroxidases were blocked with 0.3% v/v hydrogen peroxide followed by blocking of endogenous avidin/biotin (Vectorlabs, Burlingame, USA). Sections were then incubated for 1-hour with the primary antibody (1:20 rat anti-mouse CD31, clone SZ31, Dianova GmbH, Germany). After washing, a biotinylated anti-rat secondary antibody was applied for 30 minutes (1:200 Dako, UK). ABC/HRP solutions (ABC/HRP Complex Kit, Vectorlabs, Burlingame, USA) were then applied and visualised with 3,3′-Diaminobenzidine (DAB, Dako, UK). Slides were scanned using an Aperio slide scanner (Leica Biosystems, Wetzlar, Germany) at × 20 magnification. Ten 0.25 mm² boxes were placed randomly throughout the tumour section using RandomSpot software version 6.02^{38 (link)} and the number of CD31 positive vessel were counted manually. Microvessel density was calculated as the number of CD31 positive vessels per mm².

Paws from CIA mice were fixed in 4% paraformaldehyde prior to decalcification and paraffin wax embedding for H&E staining (7 µm sections) following a standard protocol. For cathepsin K detection by immunofluorescence, sections were cleared in histoclear and rehydrated before antigen retrieval in citrate buffer for 20 min at 95°C. Sections were blocked in 10% FBS in PBS for 30 min at room temperature (RT), endogenous avidin/biotin was quenched (Vector Laboratories, UK), and sections incubated overnight at 4°C with rabbit anti-mouse cathepsin K (Abcam, UK). Sections were then incubated for 60 min at RT with biotinylated goat anti-rabbit IgG (Vector Laboratories, UK) and followed with streptavidin-conjugated Alexa Fluor 647 (Vector Laboratories, UK) for 30 min at RT. DAPI (1 µg/ml) was used as a nuclear stain (Sigma-Aldrich, UK). Sections were washed in PBS with 0.05% (v/v) Tween-20 between each incubation. Following staining, sections were dehydrated through ethanol, mounted, and cathepsin K⁺ multinucleated cells on the bone surface were enumerated using an EVOS FL Auto Cell Imaging System.