Cells were fixed in 4% formaldehyde and permeabilized in 0.5% Triton X-100 before incubation in a reaction cocktail [43 μl component D, 387 μl water, 20 μl CuSO4, 50 μl reaction buffer additive (43 μl 10× reaction buffer additive + 387 μl water) and 1.2 μl Alexa Fluor 594 dye] (Thermo Fisher Scientific) for 30 min at room temperature (RT) in dark and mounted in mounting medium with DAPI (Vector Laboratories). For high-throughput imaging, cells cultured in 96-well plates (Perkin-Elmer) were formaldehyde-fixed, permeabilized in ice-cold 100% methanol for 10 min at −20°C and labelled using an Alexa Fluor™ 647 HCS assay kit (Thermo Fisher Scientific). In both cases, cells were blocked in 5% normal goat serum and 2% BSA for 1 h, followed by incubation in primary antibody at 4°C overnight and secondary antibody for 1 h at RT in dark. Cells were counterstained in DAPI and imaged using an IN Cell Analyzer 6000 Microscope. Details of antibodies are provided in Supplementary Table S5 .
Alexa fluor 647 hcs assay kit
Manufactured by Thermo Fisher Scientific
The Alexa Fluor™ 647 HCS assay kit is a fluorescent labeling reagent used in high-content screening (HCS) applications. The kit provides a fluorescent dye that can be used to label and detect specific targets within cells or tissues.
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2 protocols using alexa fluor 647 hcs assay kit
1
Immunofluorescence Staining of Cells
2
Multiparametric Immunofluorescence Imaging
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Cells were fixed in 4% formaldehyde and permeabilised in 0.5% triton X-100 before incubation in a reaction cocktail (43 µL Component D, 387 µL water, 20 µL CuSO4, 50 µL reaction buffer additive (43 µL 10x reaction buffer additive + 387 µL water) and 1.2 µL AlexaFluor 594 dye) (ThermoFisher Scientific) for 30 minutes at RT in dark and mounted in mounting medium with DAPI (Vector laboratories). For high-throughput imaging, cells cultured in 96-well plates (Perkin Elmer) were formaldehyde-fixed, permeabilised in ice-cold 100% methanol for 10 minutes at -20°C and labelled using an Alexa Fluor™ 647 HCS assay kit (ThermoFisher Scientific). Cells were blocked in 5% normal goat serum and 2% BSA for 1 hour, followed by incubation in primary antibody at 4°C overnight and secondary antibody for 1 hour at RT in dark. Cells were counterstained in DAPI and imaged using an INCell Analyser 6000 Microscope. Details of antibodies are provided in Table S5.
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