Streptavidin solution

Amine-modified polystyrene fluorescent orange latex beads (2.0 μm) were bought from Sigma Aldrich (St. Louis, MI, USA). The microparticle solution was prepared by mixing 5 μL of the microsphere stock solution into 495 μL of DI water. Rhodamine B powder was purchased from Sigma Aldrich. Rhodamine B fluorescent dye solution was prepared by mixing 1 mg of Rhodamine B powder with 10 mL of DI water, which was vortexed for complete dissolution. The water was purified using a Millipore purification system (Bedford, MA, USA). Magnetic microparticles (5 μm) were bought from Sigma Aldrich. Streptavidin solution, QuantaRed-enhanced chemifluorescent HRP substrate, and phosphate-buffered saline (PBS) were bought from Thermo Fisher Scientific (Waltham, MA, USA). A Quantikine ELISA commercial Activin A Immunoassay Kit was purchased from R&D Systems, Inc., Minneapolis, MN, USA. Glycerol was purchased from Sigma Aldrich. Solutions of different viscosities were prepared by diluting 50.00, 100.00, 150.00, and 200.00 μL of Glycerol in DI water into a final volume of 600 μL. Solutions of 4 different viscosities of 0.00115, 0.00153, 0.00209, and 0.00297 Ns/m² (1.2 cP, 1.5 cP, 2.1 cP, and 3.0 cP, respectively) were made for the viscosity experiment.

Tibiae from naïve and 5TGM1-tumour bearing mice were fixed in 4% paraformaldehyde, decalcified, and paraffin embedded bone sections were cut. Antigen retrieval was done using 1:3 dilution of trypsin enzyme for 10 min. Sections were quenched with 10% H₂O₂ and blocked with 10% goat serum solution (Invitrogen) for 30 min before incubation with rabbit anti-Sostdc1 antibody (1 μg/ml anti-Sostdc1 rabbit polyclonal antibody, Abcam) over night at 4 °C followed by goat anti-rabbit biotinylated antibody (2 μg/ml goat anti-rabbit polyclonal biotinylated antibody, R&D Systems) for 30 min and incubation with streptavidin solution (3 μg/ml, ThermoFisher) for 30 min. Antibody-antigen specific staining was developed with DAB Chromogen kit (Vector Labs, UK). Tissue sections were counterstained with Gills haematoxylin and images captured using an Aperio® ScanScope slide scanner.