TMJs were fixed in 4% paraformaldehyde, decalcified with 4% EDTA for 4 weeks, dehydrated in ethanol, embedded in paraffin, and cut sagittally into 5μm-thick sections. The most central sagittal sections of each joint were selected for safranin O staining (S2255, Sigma-Aldrich) and immunohistochemical detection with antibodies for CaSR (2 μg/ml, ab19347, Abcam), PTH/PTHrP receptor 1 (PPR) (2 μg/ml, ab7150, Abcam) and type X collagen (Col-X) (2 μg/ml, ab58632, Abcam). For negative controls, non-immune goat serum was substituted for the primary antibody.
To quantitate the morphological changes, images of safranin O-stained condylar cartilage was equally divided into three sections (anterior, middle and posterior). A region of interest (ROI) with a width of 200 μm for rat and 70 μm for mouse was placed at the center of each section and an OA grade (0-VI) was assigned based on the previously reported OARSI score system (25 (link)) to indicate the degree of TMJ OA progression (26 (link)). The values from three ROIs was averaged and reported for each sample.
To quantitate the morphological changes, images of safranin O-stained condylar cartilage was equally divided into three sections (anterior, middle and posterior). A region of interest (ROI) with a width of 200 μm for rat and 70 μm for mouse was placed at the center of each section and an OA grade (0-VI) was assigned based on the previously reported OARSI score system (25 (link)) to indicate the degree of TMJ OA progression (26 (link)). The values from three ROIs was averaged and reported for each sample.