Most commercial fluorescent markers fail in reporting endocytosis in IHCs.

<p>(<b>A</b>) FM 1-43 labels IHCs strongly in conditions where endocytosis is strongly reduced by low temperature (on ice, middle panel) or abolished by fixation (right panel). The labeling pattern is identical to that obtained at room temperature (RT, left panel). (<b>B</b>) Styryl dyes from the FM family (FM 1-84, FM 4-64, FM 4-64FX, AM 1-43), ranging in sizes between 450 and 470 Da, permeated IHCs in a similar fashion to FM 1-43 at room temperature. In contrast, the larger FM 3-25 (843 Da) did not penetrate into the tissue, producing a very faint labeling. The membrane-binding dye 5-dodecanoylaminofluorescein (DCF, 530 Da) had a similar effect. (<b>C</b>) An additional membrane dye, Di-2-ANEPEQ (549 Da), labeled IHCs in the same fashion as FM 1-43. Soluble compounds, such as calcein (623 Da) and 3000 Da Dextran coupled to fluorescein, labeled the intercellular spaces throughout the organ of Corti, but were not taken up efficiently by the cells. All dyes were used at a concentration of 10 µM, with the exception of Di-2-ANEPEQ, which was used at 100 µM, and DCF, used at 180 µM. Scale bars 10 µm. (<b>D</b>) Analysis of labeling intensity for all dyes that appeared to penetrate into the IHCs. The fluorescence intensity of the cells (excluding the nucleus) was normalized to the background intensity, and is expressed as fold over background. The black bars show results from cells incubated with the different dyes at room temperature. The gray bars show cells incubated on ice, at 2–4°C. The following numbers of IHCs were analyzed. FM 1-43: 26 at room temperature, 19 on ice; FM 1-84: 7, 27; FM 4-64: 13, 23; FM 4-64FX: 8, 20; AM 1-43: 20, 16; DCF: 8, 15; Di-2-ANEPEQ: 30, 35.