[Histonet] problem with melanin staining reddish-brown byAEC chromogen

[Katri Tuomala]

Carla,
We actually choose to use AEC chromogen on human melanoma cases, rather than 
DAB. I don't know about fish, but human melanin is naturally brownish black, 
not red, so AEC staining is easy to pick out. Dab is not good. I have tried 
to do melanin bleach step with potassium permanganate before IHC, but it can 
diminish the antigenic reactivity and you may end up with false negative 
result.
Katri


Katri Tuomala
Hamilton, Ontario, Canada


----- Original Message ----- 
From: "Jennifer MacDonald" <JMacDonald <@t> mtsac.edu>
To: "Carla M Conway" <cmconway <@t> usgs.gov>
Cc: <Histonet <@t> lists.utsouthwestern.edu>; 
<histonet-bounces <@t> lists.utsouthwestern.edu>
Sent: Wednesday, March 23, 2005 6:56 PM
Subject: Re: [Histonet] problem with melanin staining reddish-brown byAEC 
chromogen


> What about bleaching the melanin?
>
>
>
>
>
> Carla M Conway <cmconway <@t> usgs.gov>
> Sent by: histonet-bounces <@t> lists.utsouthwestern.edu
> 03/23/2005 02:48 PM
>
> To
> Histonet <@t> lists.utsouthwestern.edu
> cc
>
> Subject
> [Histonet] problem with melanin staining reddish-brown by AEC   chromogen
>
>
>
>
>
>
> Hello all,
>
> I am optimizing my protocol for a new antibody against the nucleocapsid
> protein of a fish rhabdovirus. Unfortunately, melanin pigment (especially
> large concentrations of it) is reddish-brown after staining with the AEC
> chromogen. This occurs on controls and test tissue, regardless of  primary
> antibody dilution. Determining actual positive areas (if any) is
> problematic. I am using Dako's EnVision+ system and have been pleased with
> results using other antibodies. Switching to DAB then staining with Azure
> B
> is an option (this system uses either AEC or DAB), but I prefer to stay
> with AEC if at all possible. Comparing an H&E stained slide to its IHC
> counterpart is time consuming, but helpful. I have thought about reducing
> the chromogen incubation time from 10 min to 5, but otherwise am at a loss
> for ideas.
>
> Any and all suggestions will be greatly appreciated.
> Also, thanks so much for your answers to my recent questions.
>
> Sincerely,
>
> Carla Conway
> Western Fisheries Research Center
> 6505 NE 65th St
> Seattle, WA 98115
> ph: 206-526-6282
> fax: 206-526-6654
>
>
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