Detecting Telomeric APBs by IF/FISH

IF/FISH experiments were carried out as described (Zeng et al, 2018 (link)) using antibodies against SLX4IP (Sigma-Aldrich), PML (Santa Cruz Biotechnology), or γH2AX (Cell Signaling Technology) combined with a telomere leading strand probe [5′-(CCCTAA)₃-3′] conjugated to cyanine-5 (PNA Bio). Fluorescence detection was accomplished using a secondary antibody conjugated to Alexa Fluor 488 (Invitrogen). Images were captured using a Leica TCS SP8 STED confocal microscope (Light Microscopy Imaging Core, CWRU). The cells were classified as APB⁺ according to (Fasching et al, 2007 (link)).

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Robinson N.J., Morrison-Smith C.D., Gooding A.J., Schiemann B.J., Jackson M.W., Taylor D.J, & Schiemann W.P. (2020). SLX4IP and telomere dynamics dictate breast cancer metastasis and therapeutic responsiveness. Life Science Alliance, 3(4), e201900427.

Publication 2020

SLX4IP γH2AX Alexa Fluor 488

Alexa fluor 488 Antibodies Antibody Cells Confocal microscope Fish Fluorescence Light microscopy Telomere

Corresponding Organization : Case Western Reserve University

Other organizations : University School

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Variable analysis

independent variables

Antibodies against SLX4IP (Sigma-Aldrich), PML (Santa Cruz Biotechnology), or γH2AX (Cell Signaling Technology)

dependent variables

Fluorescence detection using a secondary antibody conjugated to Alexa Fluor 488 (Invitrogen)
Classification of cells as APB+ according to (Fasching et al, 2007)

control variables

Telomere leading strand probe [5′-(CCCTAA)₃-3′] conjugated to cyanine-5 (PNA Bio)
Leica TCS SP8 STED confocal microscope (Light Microscopy Imaging Core, CWRU)

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