FRET-CLSM and double-labeling indirect immunofluorescence to detect close association of proteins in tissue sections

It is pivotal to identify protein-protein interaction in situ to understand protein function. Conventional methods to determine the interaction of proteins destruct tissue or are applicable to cell culture only. To identify association of proteins in cells in tissue, we adapted indirect double-labeling immunofluorescence and combined it with conventional confocal laser scanning microscopy (CLSM) to measure fluorescence resonance energy transfer (FRET). As a model system, we chose caveolin-1α and caveolin-2, two major components of endothelial caveolae, and examined their interaction in the endothelium of vessels in fixed tissues of laboratory animals and human glomus tumors. Several methodological aspects were examined. Measuring the absolute increase in fluorescence (ΔIF) was superior compared to determining the relative FRET efficiency, because it is more robust against small increases of fluorescence during measurements that results from unavoidable minimal crossreactivity of the secondary antibodies. Both, sequential and simultaneous incubation of secondary antibodies result in robust and reliable increases in ΔIF. If incubated sequentially, however, the acceptor-labeled secondary antibody should be applied first. The size of the secondary reagent (F(ab′)₂ vs whole antibody) has no major influence. In conclusion, CLSM-FRET can measure close spatial association of proteins in situ and can be applied to human surgical material.