Goat serum solution

A detailed description of this protocol can be found in [29 ]. On days 5 or 20 post eclosion, park^+/+ and park^−/− flies expressing mito-roGFP2-Orp1 or mito-roGFP2-Grx1 were anesthetized with CO₂, and brains were dissected in 2 mM N-ethylmaleimide (NEM) in 1x phosphate-buffered saline (ThermoFisher Scientific, Waltham, MA, USA). Brains were then fixed in 3.7% formaldehyde (ThermoFisher Scientific) for 15 min and washed with 0.3% Triton X-100 in phosphate-buffered saline (PBT) for 5 min four times, followed by a 30-min block in 10% goat serum solution (Invitrogen, Carlsbad, CA, USA). Brains were incubated overnight with 1.0% anti-TH antibodies purified from rabbit serum (Invitrogen, Carlsbad, CA, USA) at 4 °C. The brains were then washed four times with 0.3% PBT solution for 5 min and treated with blocking solution for 30 min. Brains were incubated with 0.5% Alexa 594 Goat Anti-Rabbit IgG secondary antibodies (Invitrogen) for two hours. After four 5-min washes in 0.3% PBS solution, brains were mounted onto microscope slides using Invitrogen™ ProLong™ Diamond Antifade Mountant (ThermoFisher Scientific) mounting media and cured for at least 30 min at ambient temperature then stored at −20 °C.

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.

BMSCs and HUVECs were cultured in osteoinductive medium and endothelial basal medium, respectively, in the presence of HPS and NHPS scaffolds, as described above. After incubating for 7 days, cells were fixed with 4% paraformaldehyde solution and washed with PBS. Next, cells were permeabilized with 0.2% (v/v) Triton X-100 for 15 min and nonspecific binding was blocked with 10% goat serum solution (Invitrogen, US). Then, BMSCs with the primary antibodies OCN (ab13420, Abcam, 1:100), OPN (ab8448, Abcam, 1:100) and HUVECs with VEGF (ab115805, Abcam, 1:100) and CD31 (ab28364, Abcam, 1:100) were incubated for 1 h at room temperature. After primary antibody incubation, cells were washed with PBS and incubated with appropriate Alexa-Fluor-coupled secondary antibodies (Life Tech, USA, 1:400) for 1 h at room temperature. The images were captured using a fluorescent microscope (ZEISS, Axio, Germany).