Ts2 inverted microscope

After each timepoint, mandibles were dissected and fixed in 10% Neutral Buffered Formalin (Sigma) for 24 h. The samples were then washed in PBS and placed in ACD Bone Decalcification Buffer (Advanced Cell Diagnostics) at 4ºC for 2 weeks, changing the solution every 2 days. Mandibles were paraffin-embedded, sectioned at 4 μm, and mounted on Superfrost glass slides (Fisher Scientific). The samples were then processed with the RNAscope^® 2.5 HD Duplex Detection Kit (Advanced Cell Diagnostics) following manufacturer’s instructions.
A total of three slides per condition (AP or contralateral control) from different samples at each time point was used for each combination of probe pairs. Brightfield images of the periapical lesion were captured at 40 × magnification using a Nikon Ts2 inverted microscope (Nikon Instruments) using NIS-elements software (Nikon Instruments). Relative gene expression within the apical osteolytic lesions was quantified using NIH ImageJ software after color thresholding. Data are presented as area of each of red and green signals over total region of interest.

Oil red O staining was used to determine the accumulation of cellular lipid droplets. After the differentiation process, the cells (2 × 10⁴ cells/well in 24-well plate) were washed with PBS (pH 7.4) and fixed with 10% w/v formalin for 15 min at room temperature. Then, oil red O solution was added to stain cellular lipid droplets for 1 h. After removal of excessive staining solution, the cells were rinsed three times with deionized water and 60% isopropanol. The stained adipocytes were observed under a Nikon Ts2 inverted microscope (Tokyo, Japan). The dye retained in the cells was extracted with 100% isopropanol, and the optical density (OD) was measured at 570 nm with a microplate reader (Anthros, Durham, NC, USA). The OD at 570 nm was calculated as a relative value compared to the total protein content (as determined by BCA assay kit) and presented as % oil red O staining.
In addition, the level of released glycerol in differentiated 3T3-L1 adipocytes was measured with a glycerol assay kit, and the amount of cellular triglyceride was determined with a triglyceride quantification kit, following manufacturer’s instructions (Sigma Aldrich, St. Louis, MO, USA). The triglyceride content was normalized with the total cellular protein content.