[Histonet] weird brownish stuff in AEC slides

[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 
methanol.

On Thu Jul 01 11:11:30 EDT 2004, Bryan Hewlett 
<bhewlett <@t> cogeco.ca> wrote:

> Rebekah,
> 
> 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
> section.
> 
> 
> 
> 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
> temperature,
> 
> 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.
> 
> 
> 
> Bryan
> 
> 
> 
> 
> 
> Reference.
> 
> 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 -----
> From: "SMITH,REBEKAH FELICIA" <rockbeki <@t> ufl.edu>
> To: <histonet <@t> lists.utsouthwestern.edu>
> 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.
>> 
>> --
>> SMITH,REBEKAH FELICIA
>> "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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> 
> 
> 
> 



--
SMITH,REBEKAH FELICIA
"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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