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.
Tcs sp5ii
Manufactured by Leica
Sourced in Germany
The TCS SP5 II is a confocal laser scanning microscope system designed for advanced imaging applications. It features a modular design, allowing for customization to meet specific research requirements. The system provides high-resolution, multi-channel fluorescence imaging capabilities.
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5 protocols using tcs sp5ii
1
Localization of Free Calcium Imaging
2
Evaluating Mitochondrial Activity in Caco-2 Cells
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JC-1 dye was used to detect the cell membrane potential and MitoTracker red dye to detect biologically active mitochondria within the cells. After intervention with ethanol and NR for 48 h, Caco-2 cells were washed with PBS and then stained with JC-1 dye (Beyotime, Shanghai, China) or MitoTracker® Red CMXRos (Thermo Fisher Scientific, Carlsbad, CA, USA) according to the instructions. The staining was immediately followed by observation using a confocal microscope (Leica TCS SP5 Ⅱ).
3
Subcellular Localization of TaPIN1 in Arabidopsis
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35S::TaPIN1-6a-CDS-GFP was inserted into the pMDC83 vector and infected into
Arabidopsis by Agrobacterium tumefaciens (strain GV3101)-mediated transformation to determine the subcellular localization of TaPIN1. Then, T1 transgenic plants were placed in hygromycin (15 mg/L) pressure medium, and after germination, 5 to 6-day-old seedling roots were excised for imaging as described [33] . Staining of roots with FM4-64 was performed as previously described [34] [35] [36] [37] [38] [39] . Confocal microscopy was performed on the root of the positive Arabidopsis plants with a Leica TCS SP5Ⅱ
(Richmond, IL, USA), and the GFP signal was observed at 505 to 530 nm emission under 488 nm excitation. Fluorescent images were captured using an LSM 880
Airyscan (Zeiss, German) with a 40× objective. Fluorescence was detected using a 488-nmbandpass filter for GFP. Images were processed using LSM image processing software (Zeiss, German).
Arabidopsis by Agrobacterium tumefaciens (strain GV3101)-mediated transformation to determine the subcellular localization of TaPIN1. Then, T1 transgenic plants were placed in hygromycin (15 mg/L) pressure medium, and after germination, 5 to 6-day-old seedling roots were excised for imaging as described [33] . Staining of roots with FM4-64 was performed as previously described [34] [35] [36] [37] [38] [39] . Confocal microscopy was performed on the root of the positive Arabidopsis plants with a Leica TCS SP5Ⅱ
(Richmond, IL, USA), and the GFP signal was observed at 505 to 530 nm emission under 488 nm excitation. Fluorescent images were captured using an LSM 880
Airyscan (Zeiss, German) with a 40× objective. Fluorescence was detected using a 488-nmbandpass filter for GFP. Images were processed using LSM image processing software (Zeiss, German).
4
UVC-Induced Autofluorescence Quantification
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In some studies where noted, after the backs and ears of C57BL/6 mouse were irradiated with UVC, the backs and ears were collected, which were imaged under a confocal fluorescence microscope (TCS SP5Ⅱ, Lecia, Wetzlar, Germany). The excitation wavelength was 488 nm and the emission wavelength was 500-550 nm.
The AF was quantified by using the following protocol: Sixteen spots with the size of approximately 100 X 100 μm 2 on the scanned images were selected randomly. The average AF intensities of the 16 spots were quantified. The average value of the 16 AF intensities was defined as the AF intensity of the sample.
The AF was quantified by using the following protocol: Sixteen spots with the size of approximately 100 X 100 μm 2 on the scanned images were selected randomly. The average AF intensities of the 16 spots were quantified. The average value of the 16 AF intensities was defined as the AF intensity of the sample.
5
Cellulose Visualization in Plant Cells
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Sections for light microscopy were stained with 0.01% (w/v) calcofluor white (fluorescent brightener 28, Sigma Chemical, St. Louis, MO) in PBS for 5 min to stain cellulose in order to visualize the cell walls present in the section, and viewed on a laser scanning confocal microscope (Leica TCS SP5Ⅱ).
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