Ultrastructural localization of blood-brain barrier-specific antibodies using immunogold-silver enhancement...

The blood-brain barrier in vivo is situated at the level of the endothelia in brain microvessels and functions to regulate the composition of the extracellular milieu of brain. In order to study the expression of barrier-specific proteins (BSPs) within the microvessels, an antiserum was prepared that reacted specifically with microvasculature in bovine brain tissue. Attempts at immunocytochemical localization of the BSP antigens on endothelia using pre-embedding and post-embedding methods with sections of bovine brain were hampered by either the poor penetration of immune reagents and/or the poor ultrastructure in preparations where fixation was light enough to preserve antigenicity. These problems were addressed by applying a method whereby the primary and secondary antisera were reacted with microvessels isolated by enzymatic digestion of bovine brain; the secondary antibody was labelled with 1 nm gold particles and, after fixation with glutaraldehyde and osmium, the size of the gold probe was amplified by a one-step silver enhancement. The microvessel pellets were then processed as for routine electron microscopy. The BSP antiserum localized to the luminal and the abluminal membranes of the endothelial cells, and there was also immunolabelling of the endothelial tight junctional area. Comparison of these preparations made with the 1 nm gold conjugates with those made with standard 5-10 nm immunogold probes demonstrates that this immunogold-silver enhancement of the 1 nm probe is a simple and more sensitive method of immunolocalization of brain capillary antigens.