Tcs sp5 2 upright microscope

To analyze the role of osteocytes in mineralization, wild-type mice were injected 5 days apart with calcein (5 mg/kg i.p, 1^st injection) and alizarin red (20 mg/kg i.p, 2^nd injection) as previously described 27 (link). Thick sections (300-400 μm) were cut from the MMA-embedded blocks of these animals with a diamond-bladed saw (Buehler, Lake Bluff, IL), ground down to a final thickness of 30-50 μm, and polished. Fluorochrome labeling was then photographed using a Leica TCS SP5-II upright microscope combined with or without DAPI staining of nuclei of osteocytes 30 (link).
For double mineral labeling, adult mice were i.p. injected with Calcein (5 mg/kg) and Alizarin Red (20 mg/kg) 5 days apart, with mice sacrificed two days after the second injection as previously described 27 (link). In the experiment with the P8 2.3 Col1-GFP mouse model, alizarin red (20 mg/kg i.p) was injected at 5 different time periods from 5 min, 15 min, 30 min, 60 min, and 4 hours before harvest to demonstrate the short-term effect of mineral deposition by osteocytes. Nondecalcified tibia samples were embedded in optimum cutting temperature compound (O.C.T. compound, Tissue-Tek) and cut into 10-μm-thick frozen sections using a transparent film as previously described 31 (link), followed by confocal imaging.