RE: enhanced DAB kits, double staining

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From:"Su, Phy-Huynh" <>
To:'Histonet' <>

I have only tried this once before, so I don't know if this can help.  I
used Vectors' DAB kit with Nickel Chloride solution in its kit.  This will
turn DAB from brown to black.  Non-specific background will be purple-ish,
and is very easily to distinguish.  I then used DAB for my second
peroxidase, because I didn't want to mess with aqueous base mounting
solution.  I used 0.5% Lithium Carbonate to really blue my hematoxylin, and
it worked nicely.  Again, I've only used this once before.  But I have used
DAB + Nickel chloride solution ever since, because of its intense color, and
easy to tell background.  

> -----Original Message-----
> From:	Gayle Callis []
> Sent:	Sunday, August 27, 2000 12:11 AM
> 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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