RE: enhanced DAB kits, double staining

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From:"Morken, Tim" <>
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We use alk phos and Fast Red almost exclusivley and use DAB as a second
chromagen in double staining. The Fast Red is a much brighter red than AEC
and contrasts much better with DAB.

Tim Morken, BA, EMT(MSA), HTL(ASCP)
Infectious Disease Pathology Activity
Centers for Disease Control and Prevention
1600 Clifton Road
Atlanta, GA 30333

PH: 404-639-3964
FAX: 404-639-3043


-----Original Message-----
From: []
Sent: Monday, August 28, 2000 3:38 PM
To: Gayle Callis
Subject: Re: enhanced DAB kits, double staining

Hi Gayle,

Have you considerd using Beta-Galactosidase as your alternate detection
system in conjunction with your HP system?  It provides a sky-blue color
and gives good contrast with Dab or AEC and allows the use of
hematoxylin for your nuclear staining.  Several companies sell
Streptavidin conjugated Beta-Galactosidase along with substrates.  I
make up my own substrate because it is less pricey.  If you're
interested I will be more than happy to post the protocol.


Gayle Callis wrote:
> We have been playing with DAB+ and AEC chromogens with double staining,
> haven't liked the color similarities (brown and red), a thing that is not
> uncommon.  Am not sure black and red is so great either, but ------
> ***One thing the researcher is insisting on IS hematoxylin
> no methyl green or nuclear fast red.
> What have people's experience with enhanced DAB kits, Zymed has one which
> gives a dark brown color (DAB plus which is their DAB = enhancer solution)
> or their DAB Black kit. Is the DAB highly sensitive, comparable to my
> beloved DAKO DAB+.
> Have been disappointed in some of the other enhanced DAB kits or too
> enthralled with some of the other HRP kits (messy background), that is
> probably my control of the situation.
> Hate giving up my beloved DAKO AEC+, unless I can postenhance this one,
> some enhancing solutions are unbelievably pricey, others more reasonable.
> Has anyone tried the Zymed enhancing solution (can be purchased
> with DAKO DAB or DAB+.  One company said theirs would work, but not
> to pay the price.
> I refuse to weigh out, mess with DAB tablets in buffers - for some of the
> metal enhancing methods, and this refusal includes Osmium tetroxide.
> Just looking for a direction on what to try first, been in the Archives on
> this,
> Guess the other question is what color combinations do you like best with
> hematoxylin counterstain, and how much interference will hematoxylin have
> with BCIP/NBT color.
> I could spend a fortune trying various companies products, but that is not
> cost effective for research dollars.
> Still reading and sleuthing this one.
> Gayle Callis
> Veterinary Molecular Biology
> Montana State University
> Bozeman MT 59717-3610
> 406 994-4705
> 406 994-4303

Hermina Borgerink BA, HT(ASCP)IHQ
Department of Pathology
Wake Forest University School of Medicine
Winston-Salem, N.C. 27157
PH (336) 716-1538
Fax (336) 716-1515

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