Figure 6.

In conventional heterologous immunohistochemistry (panel A), there is little risk that the secondary antibody will cross-react with endogenous IgG in the tissue sections (here mouse-on-rat). Thus a biotin-peroxidase approach using a biotinylated second antibody (panel B) will give rise to good specific staining (panel E). However, for homologous immunohistochemistry (here mouse-on-mouse; panel C), such secondary antibodies will also react with the endogenous IgG in the tissue, giving rise to high unspecific background staining (panel F). One way that this problem can be overcome is to use a primary antibody that has been purified and directly biotinylated (panel D), combined with a heterologous goat-antibiotin antibody as secondary antibody. This would then be detected using a conventional conjugated antigoat tertiary antibody, to give rise to an image (panel G) little different from the heterologous situation (cf. panel E). Ab, antibody.