Computational protocol: Dynamics of Brassinosteroid Response Modulated by Negative Regulator LIC in Rice

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Protocol publication

[…] GFP fluorescence was visualized under a confocal microscope (Zeiss LSM510 META, Germany) equipped with an argon laser (488 nm). GFP was excited by an Argon laser at 488 nm, and images were acquired using a 512b Roper Cascade EMCCD camera and MetaMorph software (Molecular Devices, Sunnyvale, CA). Images of LIC-, LICm-, and LICp-GFP were obtained with identical image acquisition settings. A series of images at different points along the z-axis were collected from the top to the bottom. Projection of the z-series of images results in a 3D view of the cell. To quantify the effect of 24-eBL on OsLIC-GFP localization in the time-course experiment, images were obtained with a 500-ms exposure time. Quantification of the fluorescent protein signal involved use of ImageJ (http://rsb.info.nih.gov/ij). To measure the ratio of nuclear to cytoplasmic signals (N/C ratio) for LIC-GFP for each cell, small areas were drawn, and measurements of integrated densities were taken from representative areas within the nucleus, cytoplasm, and background (central vacuole) of each cell. Each sample of at least 20 cells was measured 3 times; the average N/C ratio were then calculated . […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Arabidopsis thaliana, Bovine orthopneumovirus, Oryza sativa
Diseases Hypertension