Zen 2012 lite imaging software

All imaging examinations were performed with an LSM 700 confocal microscope (Carl Zeiss AG) at room temperature (22–25℃). The external solution for confocal imaging contained 160 mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 8 mM glucose, adjusted to pH 7.4 with NaOH and an osmolarity of 321–350 mOsm. For live-cell imaging, images were obtained by scanning cells with a ×40 (water) apochromatic objective lens at 1024 × 1024 pixels using digital zoom. Analysis of line scanning of fluorescence images was performed using the ‘profile’ tool in Zen 2012 lite imaging software (Carl Zeiss Microimaging). To analyze colocalization, we performed quantitative colocalization analysis using Fiji software with the Colocalization Threshold plugin to determine the Pearson’s correlation coefficient (R). Pixel intensities were presented as 2D intensity histograms with a linear regression line and as bar graphs with mean R values. All images were transferred from LSM4 to JPEG format.

TsA201 cells were imaged 1–2 days after transfection on poly-L-lysine coated chips with a Carl Zeiss LSM 700 confocal microscope (Carl Zeiss AG) at room temperature. The external Ringer’s solution contained 160 mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 8 mM Glucose, adjusted to pH 7.4 with NaOH. For time courses, cell images were scanned with a 40 X (water) objective lens at 512 X 512 pixels using digital zoom. During time course experiments, images were taken every 5 s. Quantitative analysis of the cytosolic fluorescence intensity was performed using the ‘measure’ tool for the region of interest in ZEN 2012 lite imaging software (Carl Zeiss MicroImaging). All confocal images were transferred from LSM5 to JPEG format, and raw data from time courses was processed with Microsoft Office Excel 2012 (Microsoft) and Igor Pro (WaveMetrics, Inc.).