Envision dual link system hrp rabbit mouse kit

After necropsy, samples were immediately fixed in 4% paraformaldehyde (Sigma-Aldrich) overnight and then transferred to 80% v/v ethanol until further analysis. Bone samples were decalcified for up to 5 weeks in 10% EDTA (pH 7.4) at 37 °C before all samples were subjected to routine processing and were embedded in paraffin wax. Serial sections were used for hematoxylin and eosin (H&E) staining and immunohistochemistry as outlined in Supplementary Table S6. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide (Sigma-Aldrich) for 15 min and non-specific binding sites were blocked with 2% bovine serum albumin (BSA; Sigma-Aldrich). Primary antibodies were diluted in the blocking buffer. Immunoreactivity was detected using the EnVision + Dual Link System-HRP Rabbit/Mouse kit (Dako, Glostrup, Denmark) and was color developed with liquid diaminobenzidine chromagen (Dako). Sections were counterstained with Mayer’s Hematoxylin (Sigma) before dehydration and mounting. Human tissue and murine tissue were used as positive and negative controls respectively, for validating that antibodies reacted with human and not murine tissues^{5 (link)}. Images were captured using a Leica SCN400 or a 3D Histech Panoramic high throughput slide scanner.

After necropsy, samples were immediately fixed in 4% paraformaldehyde (Sigma) overnight and then transferred to 70% (vol/vol) ethanol until further analysis. Bone samples were decalcified for up to 5 weeks in 10% EDTA (pH 7.4) before embedding in paraffin. Serial sections were used for H&E staining and immunohistochemistry as outlined in Supplementary Table S1. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide (Sigma) for 15 min and non-specific binding sites were blocked with Background Sniper (Biocare Medical, Concord, CA, USA). Primary antibodies were diluted in antibody diluent (Dako, Australia). Positive immunoreactivity was detected with the EnVision+ Dual Link System-HRP Rabbit/Mouse kit (Dako) and was developed with liquid diaminobenzidine chromogen (Dako). Sections were counterstained with Mayer’s Hematoxylin (Sigma) before dehydration and mounting. Human murine tissues were used as positive and negative controls respectively, for human-specific antibodies (Supplementary Figure S3). Images were captured using a Leica SCN400 high-throughput slide scanner.