Indirect immunolabeling

Brown JK, Pemberton AD, Wright SH, Miller HRP (2004) Primary antibody Fab fragment complexes a flexible alternative to traditional direct and indirect immunolabeling techniques. J Histochem Cytochem 52 1219 1230... 

For indirect immunolabeling, the antibody of desired specificity, referred to as the primary antibody, is applied first, without fluorochrome conjugation. After excess primary antibody is washed away, a fluorochrome-conjugated, second antibody is introduced. Antigenic specificity of the second antibody is to immunoglobulin... 

Provided that monospecific antibodies are used, the precision of protein localization is primarily determined by the size of the immunoglobulin and protein A/G molecules and the diameter of the gold particles. Precision therefore depends on whether the direct or indirect immunolabeling method is applied and, in the latter case, whether a secondary antibody or protein A/G is used. The different cases have been analyzed in detail by Kellenberger and Hayat (1991), who estimated that the direct method yields a precision of 8-10 nm whereas the... 

Indirect immunoenzyme procedures. Reasonably rapid. Permanent preparations. Simple nuclear counterstains may be used for studying tissue architecture in relation to immunolabeling. Use of toxic chromogens is involved. Need to block endogenous enzyme activity. 

In general, the accompanying two-step indirect methods provide sufficient sensitivity to successfully complete double/ triple immunolabeling procedures in 1 day. 

In mammalian cells transfected with a luciferase-expressing plasmid, we showed by indirect immunofluorescence that luciferase is localized to small vesicular structures (24). Subsequent work involving double-immunolabeling experiments revealed that these vesicles were peroxisomes (37). Interestingly, luciferase is also peroxisomal in the firefly lantern, in yeast and in plant cells... 

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