Computational protocol: CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex

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Protocol publication

[…] Images of cells were collected with a DeltaVision optical sectioning system using PlanApo 100×/1.40 NA oil, PlanApo 60×/1.40 NA oil ph3 or UPlanApo 20×/0.70 NA dry objectives (Olympus), using a cooled CCD camera (Series300 CH350; Photometrics). Fluorescence signals were visualized using a quad filter set (86000; Chroma Technology Corp.) for multiple color imaging, or Endow GFP bandpass emission filter set (41017; Chroma Technology Corp.) for GFP imaging. The out-of-focus signals were removed using the deconvolution technique with the DeltaVision system. Confocal imaging was performed using LSM510 confocal laser scanning microscope (v. 2.3; Carl Zeiss MicroImaging, Inc.). TIRF microscopy was performed on an Olympus IX70 (PlanApo 100×/1.45 NA, oil TIRFM objective), equipped with a 488-nm argon laser line (MELLES GRIOT), an objective-type TIRF illuminator (Olympus), and an OrcaER cooled CCD camera (Hamamatsu Photonics). The system was controlled by Aquacosmos software (Hamamatsu Photonics). The quantification and analysis of fluorescent signals was performed using MetaMorph software (Universal Imaging Corp.). Images were prepared for publication using Photoshop (Adobe). Statistical analysis was performed using with a SigmaPlot (SPSS Inc.) and Statistica for Windows (StatSoft Inc.). Unless stated differently, the statistical significance of the observed differences was evaluated using Kolmogorov-Smirnov two-sample test and in all plots SD is indicated. For FACS analysis, suspended cells were fixed in cold ethanol, labeled with anti-CLASP1/2 antibodies and Cy5-conjugated secondary antibody, and analyzed using FACSCalibur (Becton Dickinson). […]

Pipeline specifications

Software tools MetaMorph, SigmaPlot, Statistica
Applications Miscellaneous, Conventional fluorescence microscopy
Organisms Homo sapiens