Telomere Damage Assessment via γH2A.X

Sections of paraffin-embedded liver were mounted on glass slides and processed for γH2A.X staining on telomere regions as previously described (43 (link)). γH2A.X antibody was purchased from Cell Signaling. Biotinylated secondary antibody and Fluorescein Avidin DCS were purchased from Vector Laboratories. Following γH2A.X immunofluorescence, telomere immunoFISH was performed using Cy-3–labeled telomere-specific (CCCTAA) peptide nucleic acid probe (Panagene, Daejeon, KR). Counter-staining was done with DAPI and images were taken and in-depth Z stacking was used (a minimum of 40 optical slices with ×63 objective).

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Stout M.B., Steyn F.J., Jurczak M.J., Camporez J.P., Zhu Y., Hawse J.R., Jurk D., Palmer A.K., Xu M., Pirtskhalava T., Evans G.L., de Souza Santos R., Frank A.P., White T.A., Monroe D.G., Singh R.J., Casaclang-Verzosa G., Miller J.D., Clegg D.J., LeBrasseur N.K., von Zglinicki T., Shulman G.I., Tchkonia T, & Kirkland J.L. (2016). 17α-Estradiol Alleviates Age-related Metabolic and Inflammatory Dysfunction in Male Mice Without Inducing Feminization. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 72(1), 3-15.

Publication 2016

γH2A.X antibody Fluorescein Avidin DCS

Antibody Dapi Embedded paraffin Fluorescein avidin Immunofluorescence Liver Minimum Nucleic acid probe Peptide Telomere Vector

Corresponding Organization :

Other organizations : Mayo Clinic in Florida, University of Queensland, University of Pittsburgh, Yale University, Mayo Clinic, University of Newcastle Australia, Newcastle University, Cedars-Sinai Medical Center, Howard Hughes Medical Institute

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Variable analysis

independent variables

Sections of paraffin-embedded liver

dependent variables

γH2A.X staining on telomere regions

control variables

Sections of paraffin-embedded liver
γH2A.X antibody purchased from Cell Signaling
Biotinylated secondary antibody and Fluorescein Avidin DCS purchased from Vector Laboratories
Cy-3–labeled telomere-specific (CCCTAA) peptide nucleic acid probe (Panagene, Daejeon, KR)
DAPI counter-staining
In-depth Z stacking with a minimum of 40 optical slices and ×63 objective

positive controls

None specified

negative controls

None specified

Annotations

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