[Histonet] RE: double immunoenzymatic methods

[C.M. van der Loos]

Hi Kim,The best shot I can offer you is trying the method we published in JOH last September. PDF is attached with mail to your work address. That method is based on two AP staining methods with an additional HIER step in between. The second HIER step destroys/elutes the antibodies used in the first staining sequence. That prevents unwanted cross-reactions with the second staining sequence. The red-blue color combination allows to observe co-localization in a purple-blue mixed-color. Main drawback is the blue reaction product: it's a bit fuzzy compared to DAB reaction product. Take care to adapt the dilutions of your primaries, because the staining efficiency of the red and blue reaction products might be different from DAB in black. Good luck!Cheers,ChrisChris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands Date: Wed, 07 Jan 2009 12:12:05 +1100
From: Kim O'Sullivan <Kim.Osullivan <@t> med.monash.edu.au>
Subject: [Histonet] double immunoenzymatic methods
To: histonet <@t> lists.utsouthwestern.edu

Hi,

Have been having trouble getting an antibody to work for double immunofluoresecence (with the confocal microscope), but am able to get it to work on FFPE tissue with DAB black.  Does anyone out there have a method I can use that will show colocalisation using enzyme labelling methods, ie which colour choices do you make to show double labelling within the same structure I understand this is not the preferred method.

Regards

Kim O'Sullivan