Multiple labeling techniques for fluorescence microscopy

As described in Chapter 2 by Brooks, it has long been possible to localize antigens immunocytochemically using specific antibodies in conjunction with a label that is visible microscopically. Although much information can be derived by localizing a single protein/peptide, it is often useful to label simultaneously for two or more antigens within the same cells or tissue sections. There are a number of occasions when such multiple labeling techniques can be used: (1) to phenotype cells, for which no specific marker is available, using an appropriate panel of antibodies; (2) to identify which cells in a tissue or culture express an antigen of interest, by simultaneously labeling with antibodies to both this antigen and to a known phenotype marker; (3) to identify the distribution of an antigen at the subcellular level by simultaneously labeling with antibodies to both this and a known organelle marker; (4) to investigate whether several antigens of interest are colocalized, either at the cellular or the subcellular level. Although it is possible to directly label a primary antibody with a fluorochrome (direct immunofluorescence), the overall fluorescence signal achieved using this technique is often weak (2). Indirect immunofluorescence involves the use of secondary antibodies conjugated to different fluorochromes (2). This approach has the advantage that multiple secondary antibodies can bind to each primary antibody, resulting in an amplification of the signal. The most basic form of multiple labeling involves the simultaneous use of two or more primary antibodies that have been raised in different species of animals.