Double immunostaining: Good chromogen pair?

Henk Veldman H.Veldman-2 at
Fri Oct 26 06:40:45 EST 2001

Erkki Kuusisto schreef:

> Hello all,
> I would like to double stain two overlapping protein antigens on human
> tissue (paraffin sections) to show that they colocalize in the same part
> of the cell. The problem is the overlap of the antigens since I cannot use
> fluorescence.
> In most usual pairs of chromogens (e.g. DAB + NBT/BCIP), it
> is difficult to clearly show colocalization since the color products are
> so intense that one of them tends to mask the other.
> Would anyone know of a suitable chromogen pair? That is, easily
> distinguishable from each other as well as from the color resulting
> from their overlap.

If you want to observe co-localization, fluorescence is best. A combination of
DAB with fluorescence will often work also. We never could make DAB work for
co-localization in a double enzym system.
However, we did have good results with a combination of Naphthol AS-BI
phosphate / Fast blue BB for acid fosfatase (fine clear blue granules) and AEC
for peroxidase (transparent bright red). Both the labels (acid phosphatase and
peroxidase) should be in the section, after which you do the stains
sequentally. Blue first, followed by red works best in our hands. Observing
co-localization (blue granules on red -> purplish) should be no problem once
you have balanced the relative intensities of the colors by varying the
incubation time ( around 30 min each). Remember, the reaction products are
soluble in organic solvents, so you will have to use an aquous mounting


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