Modified indirect immunodetection allows study of murine tissue with mouse monoclonal antibodies.
The indirect immunodetection method is powerful in detecting antigens in situ, but to date mouse monoclonal antibodies (MAbs) could not be used in immunohistochemical studies of murine tissues without severe background staining. We report here a modification of this method in which mouse MAbs are used to detect murine antigens in cryosections. Before application to the section, mouse MAbs and conjugated anti-mouse antiserum were allowed to complex in vitro. After blocking of the unbound secondary antiserum with normal mouse serum, standard immunohistochemistry was performed. Fifty percent of a randomly chosen panel of over 40 mouse MAbs recognized their antigens in our model system. Adaptation of, for example, the fixation protocol can probably even increase this number. An MAb to the intermediate filament protein desmin, staining both smooth and striated muscle, was used to demonstrate this technique in cryosections of 15-day-old mouse embryos. In contrast to standard immunohistochemistry with the same antibodies under the same conditions, background staining was completely absent with this technique. With this modification to the well-established indirect detection method, the usefulness of mouse MAbs is significantly increased.
Hierck
B P
BP
Department of Anatomy and Embryology, University of Leiden, The Netherlands.
Iperen
L V
LV
Gittenberger-De Groot
A C
AC
Poelmann
R E
RE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology; D015925:Cryopreservation; D003893:Desmin / Q000032:analysis; D004622:Embryo, Mammalian / Q000737:chemistry; D005260:Female; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D051379:Mice; D009132:Muscles / Q000737:chemistry
D000911:Antibodies, Monoclonal; D003893:Desmin