Question.
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?
Answer(s) 1.
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 antiserum.
Corazon D. Bucana, Ph.D.
Houston, Texas
(bucana[AT]audumla.mdacc.tmc.edu)
John A. Kiernan
London, Canada
(kiernan[AT]uwo.ca)
Answer 2.
[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.
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Bret Cook
Product Specialist, DAKO Corporation
(general[AT]silcom.com)