Immunoperoxidase labelling of albumin at the endothelial cell surface of frog mesenteric microvessels

Albumin was visualised at the endothelial cell surface of perfused frog mesenteric microvessels using immuno-peroxidase labelling. Vessels in the mesenteries of pithed frogs were washed free of blood and then perfused with frog Ringer solutions containing bovine serum albumin (BSA) at a concentration of 20 mg BSA ml-1, followed by a brief Ringer flush to remove excess albumin from the vessel lumen. The tissues were fixed in 1% glutaraldehyde and a double antibody labelling technique used to identify albumin within the tissue. A dense layer of peroxidase reaction product was seen, which extended 25-50 nm into the vessel lumen. It appeared as a continuous layer lining the luminal openings of interendothelial cell clefts and vesicles open to the luminal cell surface. In some vessels a more irregular layer of peroxidase labelled albumin was seen extending 150 to 200 nm into the vessel lumen, whilst in others clumps of peroxidase labelled albumin were also seen within the vessel lumen. These data offer direct evidence that BSA does interact with the endothelial cell surface of perfused frog mesenteric microvessels but suggest that a proportion is loosley or non-specifically bound to the cell surface and can be removed by a brief Ringer flush. The remainder appears more tightly bound and resistant to Ringer flush.