Re: [Histonet] weird brownish stuff in AEC slides
|From:||"SMITH,REBEKAH FELICIA" |
I will try cooling the AEC a bit. I probably am using a kinda
high concentration of eNOS antibody (10 ug antibody/ per 1ml goat
serum), but when I try lower concentrations (I've tried 5ug,
2ug,2.5 ug,.25 ug, and .025 ug), I get too little staining. There
is definitely a lot of peroxidase already present in this tissue,
so I've been blocking for 1 hour in 1:10 30% peroxide to
On Thu Jul 01 11:11:30 EDT 2004, 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
> 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
> formation and precipitation of a yellowish-green quinone di-imine
> 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
> 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
> We often see this metachromatic effect during the optimization
> process for
> new antibodies.
> Re-titration of the primary antibody to a lower concentration
> 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
> techniques. Histochemistry 1987; 86: 471-478.
> ----- Original Message -----
> From: "SMITH,REBEKAH FELICIA"
> 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
>> problem with my AEC, my hematoxylin or something else. Anyone
>> any ideas? Thanks in advance, y'all have already been really
>> SMITH,REBEKAH FELICIA
>> "You are a child of the universe, no less than the trees and the
>> You have a right to be here and whether or not it is clear to
>> no doubt the universe is unfolding as it should. Therefore be at
>> peace with G-d, whatever you conceive Him to be. And whatever
>> labors and aspirations,in the noisy confusion of life, keep peace
>> in your soul.-Max Ehrmann,"Desiderata"
>> Histonet mailing list
"You are a child of the universe, no less than the trees and the
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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