Tritc oo ttaggg 3 labeled pna probe

Cells were grown on coverslips and fixed in 2% paraformaldehyde for 5 min. Coverslips were then incubated with 500 µg/mL RNase A in blocking solution (1 mg/mL BSA, 3% goat serum, 0.1% Triton X-100, 1 mM EDTA in PBS) for 1 h at 37°C. Coverslips were dehydrated in an ethanol series (70%, 90%, and 100%) and hybridized at room temperature with a TRITC-OO[TTAGGG]3-labeled PNA probe (PNA Bio) in hybridization buffer (70% formamide, 1 mg/mL blocking reagent [Roche], 10 mM Tris-HCl at pH 7.2). Following washes, coverslips were stained with DAPI and imaged. A minimum of three independent experiments was performed for each datum shown unless indicated otherwise. For each experiment, a minimum of 100 nuclei was imaged, blinded, and scored as follows: A cell was considered positive when at least five ss-TeloC foci could be detected. To determine the number of foci/cell, at least 200 nuclei were scored blindly for the presence of ss-TeloC foci.

Immunofluorescence (IF) staining and IF-FISH were executed as described previously (Li et al. 2017 (link)). Primary antibodies used in this study were as follows: PML (Santa Cruz BIotechnology PG-M3), BLM (Bethyl A300-110A-M), Sp100 (Abcam ab43151), RPA (Thermo Scientific PA1-23299), Flag (ABM G191 and Sigma F7425), GFP (Invitrogen A6455), Myc (Cell Signaling 2276), TRF2 (Millipore 05-521 and Novus Biologicals NB110-57130), RMI1 (Novus Biologicals NB1001720), and HA (clone 16b12, Fisher Scientific NC9378714). Secondary antibodies used were Alexa 488, Alexa 555, and Alexa 647 (Molecular probes, Life Technologies). For IF-FISH, following the IF protocol, coverslips were denatured and hybridized TRITC-OO[TTAGGG]3-labeled PNA probe (PNA Bio). Digital images were taken using Zeiss Axio Imager M1. A minimum of 100 cells per condition were analyzed. A minimum of three independent experiments were performed unless otherwise indicated. Images were blinded and scored as following: Cells were defined as positive when at least five foci colocalized with telomeres.