*4.7. Fluorescence in Situ Hybridization (FISH) and Relative Telomere Length Determination*

Cells grown on coverslips were fixed with 4% paraformaldehyde in PBS 1× for 10 min, washed three times in PBS 1X and permeabilized with Triton 0.2% in PBS 1X for 12 min. Cell preparations were treated for 20 min at 37 ◦C with 100 μL of RNase (1 μg/mL), washed three times with PBS 1X and dried. Afterwards, coverslips were incubated in hybridization buffer (20 mM Na2HPO4 [Ph 7.4], 20 mM Tris [pH 7.4], 60% formamide, 10% BSA) with 1ng/μL Cy3-conjugated telomere probe (Cy3 conjugated G-strand probe [5´-GGGTTAGGGTTAGGGTTA-3´]) added. Coverslips were then incubated at 80 ◦C for 2 h in the dark for denaturation, prior to incubation at room temperature overnight for hybridization. Next day, coverslips were washed twice with SSC 2×/1% Tween 20, for 10 min at 60 ◦C; twice with SSC 1×/0.1% Tween 20 and once with SSC 0.5×/0.1% Tween 20. Finally, cells preparations were incubated for 10 min at room temperature with DAPI (0.2 μg/μL, Sigma-Aldrich Inc.) for nuclei visualization, washed with PBS 1×, and mounted on microscope slides with VectaShield (Vector Laboratories, Inc., Burlingame, CA, USA) for confocal microscopy analysis. The analysis of the number of telomere foci and its relative length of the Q-FISH technique was performed using the Find Maxima function of

ImageJ software, 1.49 version image analysis (Wayne Rasband National Institutes of Health, USA. http://imageJ.nih.gov.ij). Raw images were converted to 8-bit gray scale to set up a binary mask that allowed the analysis of fluorescence intensity of the foci within a DAPI-positive region. The relative telomere length was calculated as follows: the telomere mean intensity was divided by the sum intensity of the DAPI signal, as described previously [61].
