Quantifying Telomere Length and Telomerase Activity

After deparaffinization, tissues were postfixed in 4% formaldehyde. Tissues were incubated at 37 °C for 15 min in pepsin solution (Sigma Aldrich). The tissue sections were mounted on slides and dehydrated in ethanol. After 10 min of air drying, 0.5 mg/ml PNA probe (Panagene) were added to each slide. The slides were incubated for 3 min at 90 °C and for an additional 2 h at room temperature in the dark. Then, the slides were incubated with DAPI (Sigma Aldrich). Confocal images were acquired as stacks using a Leica SP5-MP confocal microscope and maximum projections were done with the LAS-AF software. Telomere signal intensity was quantified using Definiens software. Fifty images per sample were captured. TL values were analyzed using individual telomere spots (300,000 telomere spots per sample). The average fluorescence intensities represent the TL of each sample [15 (link)].
Telomerase activity was quantified with a modified fluorescence telomere repeat amplification assay according to the protocol of the Telomerase Activity Quantification qPCR Assay Kit (KGA1028R, Keygen).