Re: [Histonet] DAB black/brown

From:"Thomas Pier"

If contrast is what you seek, you could always do a DAB, a Vector VIP
(very intense purple), and a methyl green counter stain.  There will be
no doubt as to what is what.

Tom Pier

>>> Gayle Callis  01/16/07 10:17 AM >>>
Chris van der loos teaches (he is the master of double IHC or any
staining) that using a blue and a red gives far better contrast along
better tinctorial quality.  He also teachs that one antibody be detected

with alkaline phosphatase (develop this color first)  and the other with

peroxidase although careful requenching of peroxidase from the first 
antibody staining can be done.

AEC with Vector Blue is superb although the Vector blue is a bit less 
sensitive DAB or AEC, which means that the primary antibody titer with
chromogen may have to increase.

AEC tends to be orange-red in color, and clashes with the brown of DAB 
which is what you observed.

A better combination with your DAB would be DAKO's Permanent red which
rich reddish -pink tones that separates much better from the brown DAB
the contrast you need.    The joy of Permanent red is the sensitivity, 
maybe even more so than AEC so retitering the antibody may be needed. 
would not need to enhance the DAB to black with Permanent red.

Good luck

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717

  At 07:06 AM 1/16/2007, you wrote:
>Can anybody help?
>For our IH we're using DAB with Ni chloride to get black color
>for double stains with AEC).
>In the last period however we get also brown coloured stainings and
even in
>one run we get some slides with brown some with black color (very bad
>our double stains).
>We used new DAB, made fresh Ni chloride solution and H2O2 but nothing
>till now...we now even got some slides stained half black half brown...
>This is especially bad for our double stainings (it's very hard to
>distinguish brown from red).
>Where could be the problem?
>Thanks a lot for any suggestions!
>Peter Boor
>Peter Boor
>University Clinic of Aachen, Dept. of Nephrology and Immunology,
>30;  52074 Aachen; Germany
>Home:   Kullenhofstr. 54A, App. 549; 52074 Aachen; Germany
>Mob.: 0049 (0) 177 2345 163
>Lab: 0049 (0) 241 80 89670
>Fax: 0049 (0) 241 80 82446
>ICQ Nr.: 259 368 084
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