Osteosoft

For whole mount bone staining, P6 mice were sacrificed by cervical dislocation. After removal of skin and viscera, carcasses were fixed in 95% ethanol for 48 hours at room temperature. The samples were cleared in 2% KOH for other 24 hours. The skeletal system was stained with Alizarin red S (Fluka, 0.2% in water with 1% KOH) for 12 hours at room temperature. For cartilage staining, carcasses were stained in 0.5% Alcian Blue 8GX (Sigma, pH 2.5 in 20% acetic acid+ 80% ethanol) for 12 hours. The samples were stored in 100% glycerol for further examination.
For cartilage staining, the tibiae of 4 months old mice were isolated under the stereomicroscope and fixed in 4% paraformaldehyde in PBS (pH 7.4) at 4 °C overnight. After two 5 min washes in PBS, the tibiae were decalcified in 30 ml Osteosoft (Merck) for 8 days (with changes of Osteosoft every 2 days). Samples were then processed for paraffin embedding. Deparaffinized sections were stained in Alcian Blue 8GX for 1 hour at room temperature. Nuclear fast red stain was used for counterstaining (for 1 min).

Mice were euthanized under deep anaesthesia by intracardial perfusion with PBS followed by perfusion with 4% (w/v) paraformaldehyde (PFA) dissolved in PBS. All of the organs were removed and fixed in 4% PFA overnight. Vertebral columns, including the spinal cords, were additionally decalcified with Osteosoft (Sigma-Aldrich) for 72 h before paraffin embedding; 2-μm-thick sections were prepared. Immunohistochemistry was performed using the Leica Bond Rxm System with the Polymer Refine detection kit (Leica). A list of all of the antibodies used is provided in Supplementary Table 2. DAB was used as chromogen, and counterstaining was performed with haematoxylin. The slides were then scanned on the Leica AT2 system, and the images were analysed using QuPath v.0.3.2 (https://qupath.github.io, University of Edinburgh, Scotland).
Quantification of histological samples was performed automatically with computer-assisted algorithms provided by QuPath. To detect the total cell counts, regions of interest were annotated and analysed automatically by positive nuclear detection. All annotations were performed in a blinded manner.