[Histonet] problem with melanin staining reddish-brown by AEC chromogen

[Carla M Conway]

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