Re: [Histonet] weird brownish stuff in AEC slides

From:"Bryan Hewlett"


I believe that what you are describing is classic so-called metachromasia of
the AEC reaction product.
Metachromasia of the AEC reaction product is seen as a progressive change in
colour from the normal rose-red to red-brown, followed by brown,
yellow-brown, then brownish-green to yellowish-green.

Metachromasia occurs in areas of high enzyme density (concentration). Enzyme
density is influenced by both the local antigen density and the amount of
attached primary antibody that is the target for the detection reagents.
Therefore, the concentration of the primary antibody directly implies the
local enzyme concentration, on the basis of a given antigenic density in the

At low concentrations of peroxidase enzyme, it has been proposed1 that the
reaction ends in the formation of a stable polymeric complex of red colour
(Wursters Red), since the decay to a precipitate occurs more rapidly than
further enzymatic oxidation.

High concentrations of peroxidase accelerate the oxido-reductive process to
the extent that there is insufficient time for the stable red polymer to
form and precipitate. Instead, further rapid oxidation results in the
formation and precipitation of a yellowish-green quinone di-imine product.
Intermediate colours are a result of both reactions.

Metachromasia in high antigen density areas is more prone to occur at
temperatures above 24°C.

Slight cooling of the AEC working solution will often correct this.

If metachromasia in high antigen density areas persists despite lowering the

or if some antibodies consistently demonstrate the phenomenon, reduction of
the detection

threshold by further dilution of the primary antibody is necessary.

We often see this metachromatic effect during the optimization process for
new antibodies.

Re-titration of the primary antibody to a lower concentration always
restores the rose-red colour.



1 Koretz K, Leman J, Brandt I, Möller P: Metachromasia of
3-amino-9-ethylcarbazole (AEC) and its prevention in immunoperoxidase
techniques. Histochemistry  1987; 86: 471-478.

----- Original Message -----
Sent: Thursday, July 01, 2004 9:52 AM
Subject: [Histonet] weird brownish stuff in AEC slides

> I have yet another question, guys. I've been asking a lot of
> questions about blocking endogenous peroxidase,but now I have a
> new situation. I'm still using the ABC peroxidase method staining
> sheep placenta (formalin fixed, paraffin embedded) for eNOS and
> using AEC as a chromogen and gill's hematoxylin (blued with tap
> water and water with 2 or 3 drops of ammonia) as a counterstain.
> (btw, still blocking peroxidase with 30% peroxide in methanol,
> since my professors are away and haven't been able to order
> anything better) I have the lovely red and blue contrast in most
> parts of my slides, but I also am getting areas of brownish-black
> staining (kinda DAB colored, although I'm not using any DAB). I'm
> not exactly sure where the brown comes from this and whether its a
> problem with my AEC, my hematoxylin or something else. Anyone have
> any ideas? Thanks in advance, y'all have already been really
> helpful.
> --
> "You are a child of the universe, no less than the trees and the
> stars
> You have a right to be here and whether or not it is clear to you,
> no doubt the universe is unfolding as it should. Therefore be at
> peace with G-d, whatever you conceive Him to be. And whatever your
> labors and aspirations,in the noisy confusion of life, keep peace
> in your soul.-Max Ehrmann,"Desiderata"
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