Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Using mouse primary antibodies on mouse tissues
Using a mouse monoclonal on sections of mouse tissue often makes a strong background staining because the secondary antiserum binds to mouse immunoglobulin already present in the tissue. Is there a way to get round this difficulty?
Two published methods seem quite good for this purpose. They are very briefly summarized below. For practical details consult the original papers:
Hierck,BP; Iperen,LV; Gittenberger-de Groot,AC; Poelmann,RE (1994): Modified indirect immunodetection allows study
of murine tissue with mouse monoclonal antibodies. J. Histochem. Cytochem. 42(11, Nov), 1499-1502.
Mouse monoclonal reacted with HRP-rabbit
anti-(mouse serum); then add excess normal mouse serum &
incubate with tissue.
Lu,QL; Partridge,TA (1998): A new blocking method for application of murine monoclonal antibody to mouse
tissue sections. J. Histochem. Cytochem. 46, 977-983.
Blocking with mixture of Fab and Fc
fragments from rabbit anti-mouse antibody. (Made by papain
digestion, then more Fc added). Stops background
staining of endogenous mouse IgG by the secondary
Corazon D. Bucana, Ph.D.
John A. Kiernan
[This answer does not really explain what to do, but the advertised product might interest users of mouse monoclonals.]
DAKO just released an immunostaining system for animal tissues. In particular, it excels with mouse antibodies on mouse tissue. We engage a novel technology to ensure clean background and high specificity. Stoichiometric amounts of primary-antibody complex are preformed before it is exposed to the tissue site. This
eliminates the unwanted reaction between secondary antibody and mouse tissue.
Please visit the DAKO Corporation website (www.dakousa.com) to request literature on the new DAKO ARK (Animal Research Kit). We presented a poster at the IAP meeting in Boston and this document is available by mail.
A few highlights: 1. One kit for all animal IHC testing utilizing mouse monoclonal primary Abs. 2. Use on tissue from any animal species. 3. Unique process eliminates background staining. 4. Staining results in 45 minutes. 5. Automatable
For more information please contact DAKO Technical Services at techserv[AT]dakousa.com or call 800-424-0021.
Product Specialist, DAKO Corporation