F1002

TIF assay (n = 4/group) was performed on deplasticized-MMA embedded bone sections by incubation in 0.01M citrate buffer (pH 6.0) at 95°C for 15 min then on ice for 15 min before washing in water and PBS for 5 min each. The TIF assay was performed as described.^{(3 (link))} In-depth Z-stacking was used (a minimum of 135 optical slices with ×63 objective). The number of TIFs per cell was assessed by manual quantification of partially or fully overlapping (in the same optical slice, Z) signals from the telomere probe (Cy-3-labelled telomere-specific (CCCTAA) peptide nucleic acid probe (F1002, Panagene) 54, Techno 10-ro, Yuesong-gu, Daejeon 34027, South Korea) and γ-H2AX, phosphorylation of the C-terminal end of histone H2A.X, a marker for double strand breaks in DNA (Cell Signaling Technology, Danvers, MA, USA). In z-by-z analysis. Images were deconvolved using a deconvolution algorithm, with blinded deconvolution in AutoQuant X3 (Media Cybernetics, Rockville, MD, USA). We defined a minimum of two TIFs to assign it as a TIF⁺ cell. TIF⁺ osteoblasts (~50 to 100 per bone section/animal), TIF⁺ osteocytes (~35 to 50 per bone section/animal), and TIF⁺ bone marrow cells (~1000 per bone section/animal) were quantified using FIJI (an Image J distribution software; NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/).^{(21 (link))}