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
Ms-G32
1600 Clifton Road
Atlanta, GA 30333
USA

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

email: tim9@cdc.gov



-----Original Message-----
From: hborgeri@wfubmc.edu [mailto:hborgeri@wfubmc.edu]
Sent: Monday, August 28, 2000 3:38 PM
To: Gayle Callis
Cc: histonet@pathology.swmed.edu
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.

Hermina

Gayle Callis wrote:
> 
> We have been playing with DAB+ and AEC chromogens with double staining,
but
> 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
counterstaining,
> 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
separately)
> with DAKO DAB or DAB+.  One company said theirs would work, but not
willing
> 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