Re: yellow HRP color

From:Bryan Hewlett


What you are describing, has been attributed to metachromasia of the AEC
reaction product.
This 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
This so-called metachromasia occurs in areas of high enzyme
The 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 antigen
density in the section.

At low concentrations of peroxidase enzyme, it has been proposed that the
AEC reaction ends in the formation of a stable polymeric red
complex(Wursters Red), since the formation of a precipitate occurs more
rapidly than further enzymatic oxidation. High concentrations of peroxidase,
or increased reaction temperatures, 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.

There are two courses of action to take.
First, try lowering the temperature of the substrate/chromogen to 18-20C. We
often see the effect on hot summer days!
Second, if the effect persists despite lowering the temperature, reduce the
detection threshold by further dilution of the primary antibody.
Re-titration of the primary antibody to a lower concentration always
restores the rose red colour.

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

Bryan R. Hewlett
Technical Specialist
Anatomical Pathology
Hamilton Regional Laboratory Medicine Program
Ontario, Canada

----- Original Message -----
From: "Cynthia Favara" 
To: "HistoNet Server (E-mail)" 
Sent: Monday, July 01, 2002 7:00 PM
Subject: yellow HRP color

> All,
> I just did immunohistochemisty using ascites for  PrP,Prion Protein with
> in house mouse ascites. I used a biotinylated secondary. I got positive
> staining, known because we have another in house antibody recognizing the
> same protein that was run in parallel.
> Positive staining was a yellow-brown with AEC detection. Question is has
> one noticed this color of positive staining before? If yes were you able
> tinker with the IHC and get to a normal red deposition?
> Please be gentle after all it is Monday and late in the day!!!
> Thanks to all,
> Cynthia Favara

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