A Flexible Procedure for Performing Telomere FISH and Immunofluorescence in Cells Expressing Fluorescent Proteins.

Abstract

Visualizing telomeres using fluorescence in situ hybridization (TelFISH) has long been an essential tool in telomere biology experiments. Combining TelFISH with immunofluorescence (IF) is also a well-established method (IF-TelFISH), for example, in identifying telomere dysfunction-induced foci, where telomeres co-localize with 53BP1. More recently, native telomere FISH (nTelFISH) has become an important tool for assaying Alternative Lengthening of Telomeres, a telomere maintenance mechanism used in some tumor cells. Expressing fluorescent proteins is also an essential tool in cell biology, allowing visualization of proteins and structures that may remain undetectable by other methods. Because FISH buffers denature proteins, performing the assay in cells with an expressed fluorescent protein (FP) results in loss of FP signal. A method for integrating IF and telomere FISH in cells with an expressed fluorescent protein that preserves the FP signal even after FISH is described here. Alternatives are provided for denaturing and native telomere FISH in combination with several IF and FP scenarios. Taken together, this protocol offers a flexible framework for exploring telomere biology in both normal and tumor cells.