Interaction analysis of tight junction proteins by Förster resonance energy transfer is recommended in live cells rather than after paraformaldehyde fixation.

The tight junction regulates the paracellular passage of solutes and water. It is primarily constituted by the family of claudins which assemble to huge multimers and basically seal the paracellular cleft. Certain claudins, however, can function as subunits of selective paracellular channels and thereby mediate paracellular ion and water flux across epithelia. In the thick ascending limb of Henle's loop (TAL), ion handling depends on the spatial separation of paracellular sodium and magnesium transport. Magnesium is conducted via a claudin-16/19 complex, whereas sodium permeation is enabled by claudin-10b which forms autonomous tight junctions without interacting with other TAL claudins. Förster resonance energy transfer (FRET) analysis is a well-established technique to study protein-protein interactions, based on the non-radiative energy transfer between closely positioned donor and acceptor chromophores. FRET has been broadly used to analyze claudin oligomerization as a prerequisite for tight junction formation. Although FRET can be performed in both living and fixed cells, comparability between these conditions remains poorly characterized. While fixed-cell FRET analyses are common for transmembrane ion channels, data for claudins under fixation are lacking. In this study, we evaluated the suitability of paraformaldehyde fixation for analyzing interaction between TAL claudins -10b and -19. Using acceptor photobleaching FRET, we compared the energy transfer efficiencies in live and fixed cells expressing fluorophore-tagged claudins. Fixation markedly altered energy transfer efficiency, leading to false-positive evaluation of apparent claudin interactions. These findings demonstrate that paraformaldehyde fixation compromises the accuracy of FRET measurements and indicate that FRET analyses are best performed in living cells.