Telomere Length and Senescence Assays

Telomere length was determined by quantitative telomere in situ hybridization (Q-FISH) [64 (link)] and telomere restriction fragment analysis [65 (link)] using a Cy3-labeled (CCCTAA)₃ PNA probe (Panagene) and a Telo-C-Biotin (CCCTAA)₃ probe (PNA Innovations), respectively. TIFs were detected by immunofluorescence and telomere-FISH using primary γH2AX antibody (#05-636-AF488, Millipore) and Alexa Fluor 488 dye-conjugated secondary antibody (#R37120, Invitrogen), followed by fixation and telomere-FISH [65 (link)]. TIFs were scored by the co-localization of γ-H2AX and telomere-FISH signal. β-galactosidase activity was carried out using the SPiDER-β-galactosidase staining kit (#SG04-1, Dojindo).

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Stock A.J., McDevitt R.A., Puligilla C., Wang Y., Zhang Y., Wang K., Sun C., Becker K.G., Lehrmann E., Wood WH 3.r.d., Gong Y., Aqdas M., Sung M.H., Hoffmann V., Liu C., Gorospe M., Harrington L., Ferrucci L, & Liu Y. (2022). Aberrant expression and localization of the RAP1 shelterin protein contribute to age-related phenotypes. PLOS Genetics, 18(11), e1010506.

Publication 2022

Cy3-labeled (CCCTAA)3 PNA probe Alexa Fluor 488 dye-conjugated secondary antibody SPiDER-β-galactosidase staining kit

Alexa fluor 488 Antibody Biotin Fish Immunofluorescence In situ hybridization Innovations Spider Telomere β galactosidase

Corresponding Organization : Université de Montréal

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Variable analysis

independent variables

Telomere length determination method (Q-FISH or telomere restriction fragment analysis)

dependent variables

Telomere length
Telomere-induced foci (TIFs)
β-galactosidase activity

control variables

Cy3-labeled (CCCTAA)3 PNA probe (Panagene)
Telo-C-Biotin (CCCTAA)3 probe (PNA Innovations)
Primary γH2AX antibody (#05-636-AF488, Millipore)
Alexa Fluor 488 dye-conjugated secondary antibody (#R37120, Invitrogen)
SPiDER-β-galactosidase staining kit (#SG04-1, Dojindo)

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