Telomere dysfunction Induced Foci (TIF) assay for cells
The TIF assay is based on the co-localization detection of DNA damage by an antibody against DNA damage markers, such as gamma-H2AX or 53BP1, and telomeres using a telomeric probe or an antibody against the telomeric protein such as TRF1 or TRF2. Briefly, cells are plated in chamber slides. The cells that attach to the surface are rinsed once with PBS and fixed in 4% paraformaldehyde in PBS for 10 minutes. Then, cells are washed twice with PBS and permeabilized in 0.5% Nonidet-P40 in PBS, blocked with 0.5% Bovine Serum Albumin (BSA) and 0.2% fish gelatin in PBS for 30 min. The antibodies that are against the DNA damage markers (e.g. gamma-H2AX) and the telomeric protein (e.g; TRF2) are diluted in blocking solution with appropriate percentage and this primary antibody mixture is incubated on cells for 2 hours. Following three washes with PBST (1x PBS in 0.1% Triton) and 3 washes with PBS, cells are incubated with secondary antibodies for 40 minutes, then washed six times with PBS. After drying, the slides are mounted with Vectashield mounting medium with DAPI. Images are captured with Deltavision wide-field microscope using the 60x objective, then deconvoluted using Autoquant X3. TIFs are quantified using Imaris software.
Telomere dysfunction Induced Foci (TIF) assay for tissue
Tissue sections are processed for TIF analysis by fluorescence in situ hybridization (FISH). Briefly, to deparaffinize 5 μM tissue sections, xylene (2x5 minutes), 100% ethanol (2x2 minutes), 95% ethanol (1x2 minutes), 75% ethanol (1x2 minutes), 50% ethanol (1x2 minutes) are sequentially used and then washed with tap water (2x3 minutes). Deparaffinized tissue sections are incubated in sodium citrate buffer (10 mM Na-citrate, 0.05 % Tween 20, pH=6.0) in a GE microwave oven (model 1540WW002) at power 5 (20 minutes) to retrieve antigens. After tissue sections cool down, they are rinsed with 1 x PBS for 5 minutes and then dehydrated in 95% ethanol for 3 minutes. After air-drying, denaturation is conducted with hybridization buffer containing a FITC-conjugated telomere sequence (TTAGGG)3-specific peptide nucleic acid (PNA) probe (PNA Bio, Thousand Oaks, CA), 70 % formamide, 12 mM Tris-HCl pH=8.0, 0.5 mM KCl, 1mM MgCl2, 0.08 % Triton X-100 and 0.25 % BSA for 5 minutes at 80◦C, followed by 2 hours incubation in the same buffer at 37◦C. Slides are washed sequentially with 70% formamide / 0.6 x SSC (3 x 15 minutes), 2 x SSC (1 x 15 minutes), PBS (1 x 5 minutes), PBST (PBS + 0.1 % Tween 20; 1 x 5 minutes) and incubated with blocking buffer (4 % BSA in PBST) for 30 minutes. Sections are incubated with primary antibody against DNA damage marker in blocking buffer at room temperature for 1 hour. Following 2 x 5 minutes washes with PBST, tissue sections are incubated with secondary antibody in blocking buffer at room temperature for 1 hour. Sections are washed sequentially with PBST (3 x 5 minutes) and PBS (1 x 5 minutes). The slides are mounted with DAPI mounting medium. Images are captured with a Deltavision wide-field microscope using a 100x objective, then deconvoluted using Autoquant X3. TIFs are quantified using Imaris software.