Multiple Antibodies from the Same Species

Keywords Immunohistochemistry Antibody labeling Fluorescence microscopy Fluorescent immunocytohemistry Fluorescent immunohistochemistry Indirect immunocytochemistry Immunostaining 

Experimental Design Chart for Block-Between Method. Design the 2° Antibody Control for the Same Species 

Final Procedure for Two 1° Antibody Same Species with Block-Between. 

Immunocytochemistry, DOI 10.1007/978-l-4419-1304-3 12, Springer Science-l-Business Media, LLC 2010 

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Visualizing more than one epitope on one section can be accomplished by different fluorescence labeling or different sizes of colloidal gold coupled to primary or secondary antibodies. Primary antibodies from different species and adequate secondary antibodies labeled differently can be used. In case of primary antibodies from the same species, the hapten technique can be applied. A hapten is a small molecule that can be bound to antibodies dinitrophenol and arsinilate are typically used as haptens. Again, adequate secondary antibodies labeled differently can be used (14,17,32). A collection of protocols for multiple immu-nolabeling has been described by Beesley (37). 

For multiple P antibody experiments, labeled ° antibodies from the same species can be used with no problems. 

One disadvantage of the PAP method is that the primary antibody and the PAP complex must be from the same species. To have ready-made PAP complexes from multiple species to accommodate the vast library of primary antibodies available is costly and unwieldy. In fact, a particular PAP complex may not be available for all cases of primary antibody species. Also, the sensitivity is slightly less than that obtained by other technologies. However, for reducing background and ease in problem solving, this technique is still quite effective. 

When an experiment uses multiple 1° antibodies derived from the same species, perform the incubations for the first 1° and 2° antibodies, block the remaining antibody sites, and then perform the incubations for the second 1° and 2° antibodies. This procedure consists of a series of two single U antibody indirect immunocytochemistry experiments with extensive blocking between them. The key element is the blocking steps between. In this example, cultures are incubated with the first 1° antibody set, mouse anti-Ag A and 2° antibody goat anti-mouse labeled with 488 fluorophore (Fig. 12.1a), followed by steps that block the remaining antibody sites (Fig. 12.1b, c). The incubations with the second 1° antibody set are with mouse anti-Ag B antibody and 2° antibody goat anti-mouse... 

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). 

Notes In general, both secondary antibodies should come from the same host species for double/multiple labeling. 

Further improvement of microchemical methods for proteinaceous media was based on immunological techniques. The high specificity of the antigen-antibody reaction enables the discrimination of the same protein coming from different species, or the detection of multiple antigens in the same sample. Application to the analysis of artwork has been reported in two types of immunological techniques immunofluorescence microscopy (IFM), and enzyme-linked immunosorbent assays (ELISA) [31]. 

Using 1° antibodies made in the different species is the easiest way to localize multiple proteins (Chapter 11). However, it is not possible to get all 1° antibodies needed from a different species. This is especially true because there are so many mouse monoclonal antibodies available. Eventually, an experiment will need two mouse 1° antibodies or two rabbit 1° antibodies. This chapter presents the concept of combining multiple 1° antibodies made in the same species of animals. Two different approaches include block-between method and labeled Fab procedure (Lewis et al., 1993) that is available as the commercial product, Zenon (Molecular Probes/Invitrogen). 

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