RE: enhanced DAB kits, double staining

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From:"Bennett, Catherine (Katie)" <>
To:'Gayle Callis' <>

I've been doing double staining using Vector's DAB (non-enhanced) and their
Vector Red with a Hematoxylin counterstain with very pleasing results.  In
my procedure the DAB is a cytoplasmic stain while the Vector Red is a
nuclear stain.  The Vector Red is an Alkaline Phosphatase substrate and is
really sharp - almost highlighter pink color.  Looks very good and has
enough contrast to be used for quantitative morphometry.

One note about Vector Red that I found out the hard way.  While the product
spec sheet claims it can be mounted in non-aqueous media, it can be removed
or washed away by leaving the slides in ethanol or xylene for more than 30
min.  (Don't be slow about cover-slipping those slides!)

Catherine "Katie" Bresee Bennett
Sr. Research Technologist
Lovelace Respiratory Research Institute
Albuquerque, New Mexico

> -----Original Message-----
> From:	Gayle Callis []
> Sent:	Saturday, August 26, 2000 10:11 PM
> To:
> Subject:	enhanced DAB kits, double staining
> 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

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