Localization of Free Calcium Imaging

The localization of free Ca²⁺ was analyzed by fluorescence imaging as previously described by Qiu et al. [31 (link)] with some modifications. Briefly, thin slices of flesh were collected from the outer flesh tissues of both NI-treated and control fruit using a razor blade. The flesh tissues were initially washed twice on a glass slide with PBS (pH = 7.4) buffer solution, which were loaded with Fluo-4/AM at 37 °C for 1 h and then subsequently washed three times with PBS buffer solution. The Fluo-4 fluorescence maintained on the tissue was visualized under 494 nm excitation wavelength of laser light and 516 nm long-pass emission filter using a laser scanning confocal microscope (TCSSP5Ⅱ, Leica, Wetzlar, Germany). Ten replicates were performed for each sample.