Axioplan 2ie mot microscope system

Tissue and spheroid sections were stained for hematoxylin and eosin (H&E), Alcian Blue (glycosaminoglycans), Alcian Blue/Periodic Acid – Schiff (acidic and neutral mucins) and Alizarin Red (calcium deposition) using standard histological methods. Stained sections were imaged using the Zeiss Axioplan 2IE Mot Microscope System (Carl Zeiss, Jena, Germany) and the Axiovision software (Carl Zeiss). Cell nuclei and vessel counts were performed with ImageJ/Fiji, using the automatic Particle Analysis tool or the manual Cell Counter plugin, considering only those found in the chondrium layer. For each tissue, cell nuclei were counted in three independent fields, in at least 4 sections; for vessel quantification, at least 12 sections per tissue were imaged. The average numbers of cell nuclei and vessels in each tissue was expressed per unit area. Phase contrast pictures of tissues were taken using an inverted microscope Olympus IX71 equipped ORCA-R2 digital camera (Hamamatsu Corp., Bridgewater, NJ, United States). Differential interference contrast (DIC) pictures of spheroid sections were acquired using the Zeiss Axioplan 2IE Mot Microscope System (Carl Zeiss).

Horseradish peroxidase staining was performed using the Vectastain Elite ABC kits (Vector Laboratories, Burlingame, CA, United States), according to manufacturer’s protocols. All antibodies used are listed in Supplementary Table 3. Antigen retrieval was performed via trypsin digestion (0.1% trypsin for 20 min at 37°C, followed by washes in deionized H₂O and PBS). Primary antibody was incubated overnight at 4°C, followed by incubation in 0.3% hydrogen peroxide in methanol for 30 min at room temperature and 3 × 10 min washes in 1× PBS, to inactivate endogenous peroxidases. A 1 h biotin-conjugated secondary antibodies incubation, at room temperature was followed by a 30 min incubation with ABC mixture solution containing biotinylated HRP (Vectastain Elite ABC kits, Vector Laboratories, Burlingame, CA, United States) at room temperature, and finally the color was developed using DAB (3,3′-diaminobenzidine) solution (DAB Peroxidase substrate, Vector Laboratories). Stained sections were dehydrated through ascending ethanol solutions, mounted using DPX Mounting Medium (Thermo Fisher Scientific), and imaged using the Zeiss Axioplan 2IE Mot Microscope System (Carl Zeiss) and the Axiovision software (Carl Zeiss).