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Immunofluorescence for detecting three proteins. - (May/09/2012 )

Hi All,

I am doing the co-transfection experiments with the proteins. One of my protein of interest is fused with EYFP and the other with DsRed. I am using DAPI for nuclear staining. I can perform the indirect immunofluorescence to detect the third protein (it has HA epitope tag). My question is which fluorophore will be best suited in my case? Did you have any experience in staining 3 proteins in the same cell?

I am trying to do confocal microscopy of a co-transfected cell.

Thank you very much.


3 colours (4 including DAPI) will not be a problem for a confocal microscope with the appropriate LASERS (yes, it is an acronym!) and filters. You might run into some trouble with ordinary fluorescence microscopy due to cross-talk between channels, as they are not specific enough.

I would either go for a green (e.g alexafluor 488) or a purple (e.g. alexafluor 633) labeled primary or secondary antibody, depending on which filters and LASERS your confocal has.


I would suggest you to use anti-HA with secondary Alexa633, and not 488, because this dye and eYFP are very similar. I don't have good experience with fixing GFP/YFP expressing cells and direct confocal imaging. Most fluorescence will be lost, and you may need to use an anti-YFP antibody.