Cells in optical-bottom multiwell plates were fixed with 4% formalin, incubated with 0.1% triton X-100 for 20 minutes, and washed three times with 0.1 M glycine. They were refrigerated overnight with primary antibodies against e-cadherin and vimentin (Cell Signaling EMT Duplex; Danvers, MA). After washing with PBS, cells were incubated for 1 hour with mouse or rabbit Alexa Fluor conjugated secondary antibodies (Cell Signaling; Danvers, MA). Cells were mounted with ProLong Gold Antifade reagent with DAPI (ThermoFisher Scientific Molecular Probes; Waltham, MA) and imaged after 24 hours using the same exposure times for all culture conditions on an automated Zeiss AxioObserver Z1 widefield fluorescence microscope. Images were optimized for display in figures using the Hi-Lo lookup table in ImageJ.
Emt duplex
Manufactured by Cell Signaling Technology
The EMT Duplex is a specialized laboratory equipment designed for the detection and quantification of epithelial-mesenchymal transition (EMT) markers. It functions as a multi-well plate assay, enabling the simultaneous analysis of multiple samples and EMT-related targets.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Alexa fluor conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor secondary antibody, by Cell Signaling Technology (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
2 protocols using emt duplex
1
Immunofluorescence Imaging of EMT Markers
2
EMT Protein Expression Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Formaldehyde-fixed cells in optical-bottom multiwell plates were incubated overnight at 4 °C with primary antibodies against e-cadherin and vimentin (Cell Signaling EMT Duplex; Danvers, MA). After washing with PBS, cells were incubated for 1 hour with mouse or rabbit Alexa Fluor secondary antibodies (Cell Signaling; Danvers, MA). Cells were mounted with ProLong Gold Antifade reagent containing DAPI (ThermoFisher Scientific Molecular Probes; Waltham, MA) and imaged after 24 hours using a Zeiss LSM 880 confocal microscope with the same detector settings and excitation laser power settings across groups. Images were analyzed using custom Matlab scripts where fluorescent signal for each protein was normalized to the number of cells based on DAPI-stained nuclei.
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