Immunolabeling double-labeling

For double immunolabeling with primary antibodies from the same host species, it is not necessary to resort to labeling with monovalent Fab fragments when primary antibodies from the same host species are different isotypes (subclasses) of IgG, for instance such as mouse IgGl and mouse IgG3. In these cases, isotype-specific antibodies may be used to distinguish between the two primary antibodies... 

Slot, J. W. and Geuze, H. J. (1984) Gold markers for single and double immuno-labeling of ultrathin cryosections, in Immunolabelling for Electron Microscopy (Polak, J. M. and Vamdell, I. M., eds.), Elsevier, New York, pp. 129-142. 

Fig. 6. Cellular colocalaation of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in the neostriatum. Each number indicates the same glial cell showing both GDH and GS immunoreactivities. The figure is modified from Kaneko et al. (1988a), where the method for double staining is described. Briefly, GS was immunolabeled with anti-GS rabbit serum and fluorescein-labeled anti-rabbit IgG antibody, and after blocking the sections with normal rabbit serum GDH was visualized by the immunoperoxidase method with biotinylated anti-GDH rabbit IgG and ABC.

In addition to vertical bundles of myelinated axons, the cerebral cortex of monkeys (e.g., DeFelipe et al., 1990) and of humans (e.g., del Rio and DeFelipe, 1995) also contains vertically oriented bundles of unmyelinated axons that are referred to as horsetails. These horsetails are the axonal plexuses of the inhibitory double bouquet cells and can be demonstrated in monkey neocortex by immunolabeling with antibodies to calbindin and tachykinin. As shown by DeFelipe et al. (1990), in the monkey these axonal bundles are widespread and form a regular columnar system descending from layer 2 to layers 3-5. The bundles are most evident in tangential sections taken at the level of layer 3, where they can be seen to have a center-to-center spacing of 15-30 fim. In a later study of the calbindin labeled double bouquet cells in monkey striate cortex, Peters and Sethares (1997) showed that there is one double bouquet cell, and therefore one vertically oriented double bouquet cell axonal plexus, or horsetail, per pyramidal cell module (Fig. 7). Within layer 2/3 the double bouquet axons run alongside the apical dendritic clusters, while in layer 4C they are closely associated with the vertical myelinated axonal bundles. DeFelipe et al. (1989 1990) proposed that the axon terminals of the double bouquet cell synapse with the shafts and spines of basal dendrites and oblique shafts of apical dendrites of pyramidal cells, but the exact role of these vertical bundles of inhibitory axons is not known. It is likely that they constitute a vertical inhibitory system that acts upon pyramidal cells within the minicolumns. 

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