The styryl dye, ANEPPDHQ, does not occupy the membrane that internalizes during TX100- and Ca-activated MEND in BHK cells. (A) 10 µM ANEPPDHQ was applied before MEND and removed during the induction of MEND with 150 µM TX100 and again after MEND. The apparent binding rates of ANEPPDHQ are unchanged by the removal of >70% of the cell surface by MEND, and the amount of fluorescence internalized during MEND (i.e., does not wash off) amounts to no more than 15% of the initial labeling (see horizontal gray lines). (B) 8 µM ANEPPDHQ was applied and removed before and after TX100 (150 µM)–induced MEND while imaging at bandwidths of 500–580 nm (black) and 640 nmLP (gray). The optical records are scaled. Neither the apparent binding rates of ANEPPDHQ nor its spectral properties are changed after TX100-induced MEND. (C) Same as B, using Ca influx in the presence of polyamine, here EDA, to induce MEND. Dye signals, which approach a steady state in these records, are unchanged by MEND.