RE: [Histonet] Colocalization of different fluorophores

From:Gayle Callis


It was a private answer to me and just what I needed to know.  I am happy 
to share her comments.  Enjoy!

She wrote:

We constantly do four color imaging work for various PIs here at the
Buck.  I can tell you with certainty the following results:

When staining with Alexa 350, 488, 555, and 647 (which we always have
trouble picking a color when looking at coloc of all four...)

488 and 555 = yellow
350 and 555 = purple
350 and 488 = we call cyan
350 and 488 and 555 = white

adding the fourth 647 becomes a mess as you can imagine, but we always
psuedocolor it various colors and analyze the coloc individually (for
example make 647 'green' and compare with 555, and 350; then make it
'red' and compare to 488 and 350, etc.) before making any conclusions of
all four combined.

Now that I think about it, to avoid strange color combos, you could
always psuedocolor the channels red and green for any alexa dye and keep
track of the changes, this way you are always reporting the coloc as
yellow (of course this would only work if you are looking at two
proteins at a time I guess)

Hope this helps, happy staining


>Gayle Callis HTL, HT, MT(ASCP)
>Research Histopathology Supervisor
>Veterinary Molecular Biology
>Montana State University
>Bozeman MT 59717

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