Computational protocol: Association of Rho-associated protein kinase 1 with E-cadherin complexes is mediated by p120-catenin

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Protocol publication

[…] Cells were plated on glass coverslips 2 d before treatment and processing for immunofluorescence staining. Briefly, cells were fixed in 3% paraformaldehyde for 30 min and permeabilized in PBS/0.2% Triton X-100 for 5 min. Cells were blocked with PBS containing 5% bovine serum albumin (BSA) for 10 min. Cells were incubated with the indicated primary antibodies diluted in 5% BSA for 30 min, and then with secondary antibodies for another 30 min. To stain actin, Alexa Fluor 488–conjugated phalloidin was used in place of a secondary antibody. Cells were stained with 0.5 μg/ml Hoechst dye in PBS for 1 min to stain nuclei. Coverslips with stained cells were mounted onto glass slides using Prolong Gold anti-fade reagent (Invitrogen) and imaged using a fluorescence microscope (Axio-Plan 2; Carl Zeiss, Oberkochen, Germany) with a 1.4 numerical aperture, 63× oil differential interference contrast objective. Images were acquired and processed using MetaMorph software (Molecular Devices, Sunnyvale, CA). To quantify cell–cell contact localization of ROCK1, four distinct regions of the coverslip were imaged with a 20× objective. Total cells were quantified using Hoechst-dye nuclei staining, and cells with ROCK1 localized to junctions were manually counted using ImageJ. The percent of cells with ROCK1 localized to cell–cell contacts in each field was calculated and averaged. Statistical analysis was performed using a two-tailed t test. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Chemicals Calcium