My cells are transfected with GFP plasmid. I want to detect a particular cell surface marker by immunostaining. Can I use Alexa Fluor 488 dye containing secondary antibody? Will it overlap with the GFP signal (as both will be green) to give me false positive result?
I have only Alexa Fluor 488 in my lab so can I use it? If not then please suggest me a suitable dye. Thank you.
Anything that would not interfere with your absorption/emission wavelenght. As others pointed out GFP has similar emission as Alexa488 and FITC or any other dye that appear green in epifluorescence. I see that some recomended Alexa633, which is already at the verge of the visible spectrum. So it depends on what sort of light source your microscope has. If you are using laser scanning microscopy make sure your lasre source covers those wavelength. if you are using epiflourescence and your microscope is fitted with a Hg-lamp then i would rather chose a differnent fluorophore, like Alexa546 or even Cy3 as your signal with far red labels wont be good, you're even likely not to get a signal at all. You could also go the other way and use UV label…again, check your light source if they cover UV.
Thank you Alex! If you have some reference article to distinguish between two signals then please share.
You can not use Alexa 488 or any other green dye, such as FiTC. I would suggest to use PE, PerCP, APC or Alexa dyes, 568, 633, 647 etc. If you have any of this kind of questions, you can always use spectra analyzer platforms to find out the spectral overlap of the dyes. Follow the link below. Good luck
https://www.biolegend.com/spectraanalyzer
Fluorescent dyes such as Atto dye, FITC, and DyLight dye can be used .
Good luck !
Anything that would not interfere with your absorption/emission wavelenght. As others pointed out GFP has similar emission as Alexa488 and FITC or any other dye that appear green in epifluorescence. I see that some recomended Alexa633, which is already at the verge of the visible spectrum. So it depends on what sort of light source your microscope has. If you are using laser scanning microscopy make sure your lasre source covers those wavelength. if you are using epiflourescence and your microscope is fitted with a Hg-lamp then i would rather chose a differnent fluorophore, like Alexa546 or even Cy3 as your signal with far red labels wont be good, you're even likely not to get a signal at all. You could also go the other way and use UV label…again, check your light source if they cover UV.
Agree with Anette Magnussen.
You should check your light source first. (Also detector if you are trying longer wavelength)
Chroma also has a spectra viewer
https://www.chroma.com/spectra-viewer?fluorochromes=10468%2C10504
Cy5 should be a good option. But I would suggest not to use any fluorophore in the range of (350-450 nm). In my experiment I have found it to bleach GFP signal.
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