Eclipse te 2000 inverted light microscope

Dentine slices were processed as described previously (Chellaiah et al., 2000 (link)). After processing, dentine slices were incubated overnight at 37°C in a serum-free α-MEM medium. The next day, an osteoclast suspension containing 2 × 10⁴ cells was gently added to the dentine slices. After adherence for 2 h, the culture media were replaced with serum-containing α-MEM containing RANKL with or without MSM at different concentrations (20 and 40 mM). After incubation for 48 h, dentine slices were processed and stained with acid hematoxylin (Sigma, St. Louis, MO, United States) for 6 min and washed well with water. Images of resorption pits were captured using a Nikon Eclipse TE 2000 inverted light microscope using a 20× and 40× objective (24).

UMR-106 cells seeded and incubated for 7 days in a six-well plate in the presence and absence of MSM (20 mM) were used to determine the effect of MSM on matrix mineralization. Cells without any MSM but grown in the OM were used as controls. Alizarin red S (ARS) is used to stain cells after washing with phosphate-buffered saline (PBS) three times. Absolute ethanol was used to fix the cells for 30 min at room temperature. After ethanol aspiration, 2% ARS solution was added to each well and processed as described previously (Aljohani et al., 2019 (link)). For Von Kossa staining, cells were washed with PBS three times and fixed with 10% paraformaldehyde for 10 min at room temperature. After the aspiration of fixative and washing with PBS, a 5% silver nitrate solution was used as described previously (Aljohani et al., 2019 (link)). Scanning the culture plates stained for ARS and Von Kossa was done in the scanner (EPSON Perfection V200). Nikon Eclipse TE 2000-inverted light microscope were used to obtain magnified images (10× objective).