[Histonet] weird brownish stuff in AEC slides

Tue Jul 6 22:02:55 CDT 2004

Hey all! Here's an update on the brownish stains on my aec slides. 
(Btw, I sadly don't have pictures of what I'm talking about 
because our new microscope with camera has not yet arrived.)I 
tried to dilute my enzyme conjugate by 25% in the hopes it would 
avoid the metachromasia problem, but the brown stains are still 
there (although lighter) and worse, the AEC is barely visible. I 
think those of you who suggested it could be blood in the tissue 
might have a point, but my professor is not overly fond of using 
picric acid so if any of y'all have any other ideas, that would be 
great. Also, if any of you ever do both DAB and AEC in your labs, 
is it possible that one set of DAB slides could get DAB onto AEC 
slides if the some of the reagents (like PBS) are shared? (Another 
tech was working on his slides at the same time that i was doing 
my run.)

   Thanks!

Rebekah Smith

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"