Dual enhancement of triple immunofluorescence using two antibodies from the same species.
Triple immunofluorescence method with two mouse monoclonal antibodies and another rabbit polyclonal antibody was established with catalyzed reporter deposition (CARD) amplification on thick floating sections from the rat cerebellum. One of the monoclonal antibodies (anti-calbindin), diluted maximally, probed with anti-mouse IgG-horseradish peroxidase (HRP) and amplified with Cy5-conjugated tyramide, immunolabeled cerebellar Purkinje cells and their arborization. Subsequently, a rabbit polyclonal IgG (anti-glial fibrillary acidic protein (anti-GFAP)), probed with anti-rabbit IgG-HRP, amplified with biotin-tyramide and visualized with fluorescein-isothiocyanate (FITC)-streptavidin, immunolabeled Bergmann's glia. Another mouse monoclonal IgG (anti-SNAP25), probed with anti-mouse IgG-rhodamine without CARD amplification, selectively visualized synaptic sites, because the maximal dilution of the other monoclonal antibody (anti-calbindin) was below the detection threshold of this anti-mouse IgG-rhodamine. Separation of the two signals (calbindin and SNAP25), each detected through mouse monoclonal antibody, was then based on the difference of sensitivity either with or without CARD amplification. Triple immunofluorescence is possible when just one of the three primary antibodies is from different species. Intensification of two of the three signals provides further advantages to examine immunolocalization of multiple epitopes on histological sections.
Nakamura
Ayako
A
Department of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashi-dai, Fuchu, Tokyo 183-8526, Japan.
Uchihara
Toshiki
T
D000818:Animals; D000831:Animals, Newborn; D000906:Antibodies / Q000276:immunology*; D000888:Antibodies, Anti-Idiotypic / Q000378:metabolism; D000918:Antibody Specificity; D064026:Calbindins; D002384:Catalysis; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism; D000939:Epitopes; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D007124:Immunoenzyme Techniques / Q000379:methods; D007202:Indicators and Reagents; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000276:immunology / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25
D000906:Antibodies; D000888:Antibodies, Anti-Idiotypic; D064026:Calbindins; D000939:Epitopes; D005456:Fluorescent Dyes; D005904:Glial Fibrillary Acidic Protein; D007202:Indicators and Reagents; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D064030:S100 Calcium Binding Protein G; C494209:Snap25 protein, mouse; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; C048844:anti-IgG