Dual enhancement of triple immunofluorescence using two antibodies from the same species

Abstract

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.

Introduction

Multifluorolabeling immunohistochemistry is an indispensable tool to examine spatial relationship between epitopes on histological sections. It requires a clear separation of signals and sufficient intensity of each signal. Because the signals obtained with conventional fluorochromes conjugated with secondary antibodies are not always intense enough, several signal amplification methods have been developed. Among them, catalyzed reporter deposition (CARD) method is now frequently used as one of the methods of choice (Adams, 1992, Bobrow et al., 1989). Amplification with CARD method is mediated by horseradish peroxidase (HRP), usually conjugated with secondary antibodies and reacted with tyramide in the presence of hydrogen peroxide. This yields amplified signal represented by a reporter molecule, such as biotin or fluorochrome, conjugated in advance with tyramide, that accumulates around HRP (Adams, 1992, Bobrow et al., 1989). Signal amplification with CARD method allows further dilution of the primary antibody below the threshold detectable with non-amplified conventional method using fluorochrome-labeled secondary antibodies (Kumar et al., 1999, Speel et al., 1997, van Gijlswijk et al., 1997). Hunyaday et al. (1996) first reported that a combination of this highly sensitive method with conventional fluorolabeling enabled double immunolabeling based on this difference in the detection threshold even when the two epitopes were probed with primary antibodies of the same class from the same species. This double labeling with antibodies from the same species can be combined with an additional antibody from another species without danger of cross-reaction, as we demonstrated recently (Uchihara et al., 2003). Moreover, by using tyramide-conjugated fluorochromes, it is possible to amplify two immunofluorescent signals from two antibodies from different species (Uchihara et al., 2000). Because this dual amplification allows further dilution of these two primary antibodies, it can be theoretically combined with an additional primary antibody (the third antibody) from either species. A fluorochorome-labeled secondary antibody that visualizes the third antibody is not sensitive enough to immunolabel the diluted primary antibodies that have been already visualized. Triple labeling achieved in this way, therefore, involves two antibodies from the same species, one of which should be amplified, and another antibody from different species, that can also be amplified. In this study, we used, as primary antibodies, two mouse monoclonal IgGs and a rabbit polyclonal IgG to perform triple labeling. Amplification of the signal from the rabbit polyclonal IgG and another from one of the two mouse monoclonal IgGs yielded signals intense enough to be detected in the thick sections.