Immunofluorescence staining was performed as previously described4 (link),42 (link). Briefly, cells were plated on acid washed glass coverslips in six-well culture plates for two or three days. Cells were fixed with 3% paraformaldehyde in 1X PBS for 10 min, permeabilized using 0.1% Triton for 3 min, and blocked using 10% goat serum for 1 h at room temperature (RT). Cells were incubated with anti-E-cadherin primary antibody overnight at 4C. After washing five times with 1X PBS, coverslips were incubated with DAPI (0.1 μg/ml), Dy-Alexa 488 (1:500) (Thermo Fisher Scientific) for one hour at RT. Coverslips were washed for 30 min and mounted on slides using media containing 90% glycerol and 10% 1 M Tris pH 7.5. Images were captured using a Nikon A1 laser scanning confocal microscope and a 40X Plan Fluor 1.3 NA oil objective. Images were captured with lasers at 408 and 488 nm. Individual z-stacks were obtained. Two-dimensional images were displayed using Extended Depth of Focus (EDF- Nikon NIS Elements) at all wavelengths.
Dy alexa 488
Manufactured by Thermo Fisher Scientific
Dy-Alexa 488 is a fluorescent dye conjugate. It is designed for use in various bioanalytical applications, such as flow cytometry and immunoassays. The dye is composed of a dysprosium (Dy) chelate covalently linked to the Alexa Fluor 488 fluorophore.
Automatically generated - may contain errors
3 protocols using dy alexa 488
1
Immunofluorescence Staining of E-cadherin
2
Immunostaining Procedure for Cell Cultures
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Immunostaining was performed as previously described (Abell et al., 2009 (link)). Briefly, cells were cultured on glass coverslips for two or three days. Cells were fixed with 3% paraformaldehyde in 1X PBS for 10 min and then washed three times with 1X PBS. Cells were permeabilized with 0.1% Triton for 3 min. Cells were blocked with 10% fetal bovine serum for 1 hour at RT. Coverslips were incubated with primary antibody overnight at 4C. Next, the cells were washed for 30 min and then incubated with DAPI (0.1 μg/ml), Dy-Alexa 488 (1:500) (Thermo Fisher Scientific), Alexa 594 (1:250) (Cell Signaling Technology) for one hour at RT. Coverslips were washed and mounted on slides with mounting media (90% glycerol and 10% 1mM Tris pH 7.5). Coverslips were imaged using an EVOS epifluorescence microscope or a Nikon A1 laser scanning confocal microscope.
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Immunostaining Procedure for Cell Cultures
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