Localization of Blood-Brain Barrier-Specific Antibodies with Immunogold-Silver Enhancement

Epithelial-like tight junctions in vertebrate brain capillary endothelium constitute the blood-brain barrier (BBB). The function of the BBB is in part regulated by proteins that are selectively expressed in brain capillary endothelium, and the function of brain capillary specific proteins may be elucidated with immunogold-silver staining (IGSS) techniques and with antibodies specific to BBB proteins. Three endothelial enriched proteins are described in the present studies: the GLUT1 glucose transporter isoform, the endothelial transferrin receptor, and immunoreactive brain capillary specific proteins (BSPs). Three different rabbit or mouse antibodies were used. At the light microscope level, IGSS techniques are as sensitive as avidin-biotin immunoperoxidase histochemistry. The use of gold conjugated to monoclonal antibodies specific to the transferrin receptor, in conjunction with IGSS techniques at the light microscope level, give results qualitatively similar to autoradiography, and the IGSS technique is simpler and faster than autoradiography. At the electron microscope level, IGSS techniques are advantageous in that silver staining of gold labeled sections may be used to guide thin sectioning or thin sections may undergo direct postembedding silver enhancement. Finally, 1-nm labeled secondary antibodies, IGSS techniques, and bovine brain capillaries isolated with an enzymatic homogenization technique are combined to allow unique identification of brain capillary endothelial plasma membrane proteins at the ultrastructural level.