Osteoclast Resorption Pit Analysis

Osteoclasts were replated on dentine slices for 10 h–12 h in the presence of TNF-α. Resorbed areas were scanned using Bio-Rad confocal microscopy essentially as previously described.^{47 (link)} The dentine slices mounted on No. 1 coverslips were viewed with a 40 × 0.6 NA air objective. Images were recorded in the epi-reflection mode using the 488-nm line of the argon laser in a 512 × 512-pixel format. Data collection and processing were accomplished with the Zeiss LSM 410 software package. Photomicrographs were stored in a TIFF image format. The area of the pit was determined from the free-hand traced perimeter using the LSM software Area Measurement function. Images were stored in TIF format and processed by Adobe Photoshop (Adobe Systems Inc.).^{47 (link)}