Cartilage discs were embedded in Frozen Section Medium (Richard-Allan Scientific Neg50, Thermo Scientific) and frozen in dry ice-cooled isopentane. Frozen tissue blocks were stored at −80°C. The samples were cut in 9–10 µm thick sections on a CryoStar™ NX70 Cryostat (Thermo Scientific), mounted on SuperFrost Plus Adhesion slides and stored at −80°C. Sections were immediately post-fixed for 60 s in cold 95% ethanol directly before immunostaining. Sections were immunostained for the presence of ACAN (clone 4F4; Santa Cruz; at 0.1 µg/mL), COL2 (clone II-II6B3-a; DSHB; at 2.45 µg/mL), COL1 (clone EPR7785; Abcam; at 0.8 µg/mL), COL10 (clone X53 diluted 1:200; generous gift from Prof. Klaus von der Mark). Slides were incubated with primary antibodies diluted in 1.25% BSA in PBS at 4°C overnight. Negative controls were made by omitting primary antibodies (Supplementary Figure S5 ). The secondary antibodies, goat anti-rabbit IgG conjugated to Alexa 488 and goat anti-mouse IgG conjugated to Alexa 594 (Life Technologies), were diluted 1:400. The stained sections were mounted with Fluoroshield (Sigma), containing DAPI for nuclear staining. Samples were analyzed using an upright Nikon Eclipse E600 microscope equipped with an Olympus ColorView III camera. Images were opened in the image processing software Fiji (Schindelin et al., 2012 (link)), colour (red, green, blue) was assigned to the images, and images were merged. Thickness of cartilage discs was measured on the stained sections using Fiji. Five measures from representative areas of a section from each donor were taken. Nuclei were counted on the basis of DAPI stained sections prepared for immunofluorescence analysis. 20 equally-sized fields (200 × 200 px) were counted per donor using Fiji. Sections were prepared from 1 disc per donor.
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