Telomere Length Analysis in Fibroblasts

Fibroblasts at PD10 were treated with the colcemid kit (Chromocenter, Tottori, Japan) and fixed in Carnoy’s solution following the manufacturer’s protocol. The fixed cells on coverslips were treated with ribonuclease (312-01931, Nippon Gene, Tokyo, Japan) and 0.005 % pepsin (V1959, Promega, WI, U.S.), hybridized with peptide nucleic acid oligonucleotide probes (F1002, Panagene, Daejeon, South Korea), and immuno-stained with Hoechst 33342 (H342, Dojindo, Kumamoto, Japan), according to the manufacturer’s protocols. Images were recorded using a BZ-X700 microscope (Keyence, Osaka, Japan). Quantification was performed using the Telometer (https://demarzolab.pathology.jhmi.edu/telometer/), as previously described [46 (link), 47 (link)].