Computational protocol: Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition

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Protocol publication

[…] Cells were washed twice with PBS, fixed with either −20°C methanol for 5 min at −20°C or 4% paraformaldehyde/PBS for 20 min at room temperature. Cells were washed three times with PBS and then block/extracted for 30 min at room temperature in 10% normal donkey serum (Jackson ImmunoResearch) and 0.3% Triton X-100 in PBS. Cells were incubated overnight at 4°C with primary antibodies diluted in 1% normal donkey serum and 0.05% Tween-20 in PBS. Cells were washed three times for 15 min at room temperature with 0.05% Tween-20 in PBS (PBS-T) and then incubated for 1 h at room temperature with secondary antibodies diluted as the primaries, washed twice in PBS-T and once in PBS, and then rinsed in water and mounted with ProLong Gold (Invitrogen-ThermoFisher Scientific).All images were captured with an Olympus FV1000 confocal microscope (Vanderbilt Cell Imaging Shared Resource) using a 60× Plan-Apochromat oil immersion objective with a numerical aperture (NA) of 1.45 and a 3× optical zoom using the FV1000 software. The individual images were converted into tiff files with the FV1000 software, and then Adobe Photoshop was used to produce the final figures. Mean fluorescence intensity was quantified using the JaCoP plug-in () of ImageJ (National Institutes of Health, Bethesda, MD). Quantitated intensities were compared by a Kruskal–Wallis test with post hoc analysis of significant means with Dunn’s test.Staining of the cysts was performed as for the Transwells, with the exception that all incubations, with the exception of the primary overnight incubation, were doubled in time for the fixation and the block and extraction steps and in the number of washes. The cysts were imaged on a Zeiss LSM 710 Meta Inverted confocal microscope using a 40× LD C-Apochromat water immersion lens with NA of 1.10 (Vanderbilt Cell Imaging Shared Resource). The individual images were converted into tiffs using Zeiss Zen software and converted into movies using Imaris software (Bitplane; Vanderbilt Cell Imaging Shared Resource). […]

Pipeline specifications

Software tools JACoP, ImageJ, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Canis lupus familiaris
Chemicals Calcium