Ma5 12789

Initially, the paraffin sections were removed using xylene after warming for 1 h in a wet box. Subsequently, enzymatic digestion with hyaluronidase (18240-36; Nacalai Tesque, Inc., Kyoto, Japan) was performed to expose the antigens in the specimens. Additionally, to minimize non-specific reactions, the specimens were blocked using 3% bovine serum albumin. Then, the specimens were subjected to incubation with the following primary antibodies at 4 °C: anti-ki67 (× 200, M7240, Abcam, Cambridge, United Kingdom), anti-TNAP (× 300, ab65834, Genetex), anti-ENPP1 (× 300, rabbit, Genetex), anti-Col2 (× 300, MA5-12789, Thermo Fisher, Tokyo, Japan), and anti-Col10 (× 300, GTX37732, Genetex). Finally, the specimens were further incubated with secondary antibodies for 30 min at 25 °C and then mounted with a DAPI-containing mountant for fluorescence observation (Abcam). The histological analysis, specifically the counting of Ki67-positive cells, was performed on samples from three rats, with all positive cells counted through visual inspection. Furthermore, standardized exposure times of 250 ms for Ki67 and 3 ms for DAPI were applied, and the enumeration of positive cells was performed visually. For light observations, TNAP and ENPP1 were combined with diaminobenzidine.

Embryos were collected and fixed in 4% PFA for 2–5 h at 4°C. Samples were then washed in PBS at 4°C for 1 h before being placed into 30% sucrose 4°C overnight to eliminate the remaining PFA. Tissues were subsequently embedded in OCT (Tissue-Tek #25608-930) and sectioned into 30–50 μm-thick sections at −20°C. Sections were equilibrated in PBS at room temperature prior to imaging. Primary antibodies (anti-Sox9, Sigma #HPA001758; anti-Col2, Invitrogen #MA5-12789) were applied to the sections for overnight incubation at 4°C. Samples were washed in PBST prior to incubation in secondary fluorescent antibodies for 1 h at room temperature. Images were acquired with an LSM880 confocal microscope.