[Histonet] Double Immunohistochemical staining

Gayle Callis gcallis <@t> montana.edu
Wed Oct 25 12:57:57 CDT 2006

Dear Liz and others,

Chris van der Loos has always prefered using a peroxidase system AEC 
chromogen with alkaline phosphatase Vector blue to see purple 
colocalization.   Liz is correct on the DAB/AEC issue from his book, and 
her use of DAB with Fast red is ideal.

DAB combined with AEC in general is a poorer color combination.  AEC is an 
orangish red and DAB is brown and contrast of these colors can be less 
appealing than using a red chromogen with magenta red - pinkish red tones 
to give better contrast and color separation.   Orange red fights with the 
brown and we didn't like it.  An alkaline phosphatase method using any of 
these chromogens (new fuchsin, fast red or very sensitive Permanent red (a 
van der Loos favorite))  with brownish DAB will avoid the clash of colors 
with AEC.  Enhanced DAB could be tried if one wants a black chromogen, or 
even a darker brown with new DAB enhancers.

He also prefers to use one alkaline phosphatase method and then a 
peroxidase method.   If one uses two peroxidase methods, then another 
peroxidase block should be done between the two HRP steps to ensure 
quenching of first HRP enzyme before using another HRP in with second 
antibody.  This is not necessary for alk phos and then peroxidase.

  Liz wrote:

If I remember correctly from Chris van der Loos lecture and his book on
multiple staining methods if the two antigens are co-localized then DAB is
not a suitable chromagen. If the antigens are not co-localized then DAB
would be fine.  Please feel free to correct me if I'm remembering this
wrong. I commonly use DAB with Fast Red for antigens that will stain
different cells or different cellular components.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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