Multiple Immunofluorescence Staining

Whereas multicolor immunoenzyme staining is applicable only for separately located antigens (see Chap. 7), multicolor fluorescence immmunostaining makes it possible to colocalize antigens not only in the same cell but also in the same cellular compartment. Simultaneous immunolocalization of antigens using fluorescent antibodies can be fulfilled both by the direct (see Sect. 4.1) and indirect (see Sect. 4.2) methods. With the direct method, primary antibodies are labeled with fluorescent dyes, while with the indirect method, primary antibodies are applied as unlabeled antibodies and the visualization is performed with secondary antibodies that are labeled with fluorescent dyes. 

Indirect methods for immunofluorescent detection of multiple tissue antigens in their simplest form make use of primary antibodies that are raised in different species and accordingly can be visualized with differently labeled species-specific secondary antibodies (see Sect. 8.1). However, quite often the appropriate combination of primary antibodies from different host species is not available. A general problem relates to the fact that the available primary antibodies may originate only from one species either rabbit or mouse. When primary antibodies are raised in the same host species, the secondary species-specific antibodies can cross-react with each of the primary antibodies (Ino 2004). 

However, if primary antibodies of the same species belong to different IgG isotypes, they can be selectively detected with secondary antibodies directed against the corresponding isotype (see Sect. 8.2). If the primary antibodies are of the same species and of the same IgG isotype, they can be modified via their haptenylation. Haptenylated primary antibodies are subsequently visualized with the use of secondary antibodies recognizing the corresponding hapten (see Sect. 8.3). 

The cross-reaction of secondary species-specific antibodies with primary antibodies from the same species is obviously avoided by direct (one antibody layer) methods. The direct method offers an easy way for simultaneous labeling of a pair or more antigens, even when using primary antibodies from the same species. Recently, a direct technique with primary antibodies that are covalently labeled by different fluorophores was described for a simultaneous detection of up to seven 

Buchwalow and W. Bdcker, Immunohistochemistry Basics and Methods, DOI 10.1007/978 3 642 04609 4 8, Springer Verlag Berlin Heidelberg 2010 

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Fig. 8.5 Schematic presentation of indirect multiple immunofluorescence staining using hapteny lated primary antibodies (AB) raised in the same host species...

As stated above, this approach is applicable only when primary antibodies are raised in different species. For double or multiple indirect immunofluorescence staining with primary antibodies raised in the same species, see Sects. 8.2. and 8.3 below. 

Advantages of direct immunofluorescence include shorter sample staining times and simpler dual and triple labeling procedures. In cases where one has multiple antibodies raised in the same species, for example two mouse monoclonals, a direct labeling may be necessary. 

In immunofluorescence microscopy, specific proteins and organelles in fixed cells are stained with fluorescence-labeled monoclonal antibodies. Multiple proteins can be localized in the same sample by staining with antibodies labeled with different fluorochromes. 

Fig. 3 Actin cytoskeleton and focal adhesion. F-actins were visualized using rhodamine phalloidin (red), and vinculins found in focal adhesions were stained using fluorescently conjugated antibodies. (A) Immunofluorescent images of a thin filamentous actin meshwork and vinculins showed that osteoblasts contain fewer and smaller focal adhesions. (B) In contrast, hMSCs showed thick actin stress fibers, and multiple and large adhesion contacts.

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