[Perfusion method of fixing and incubating alkaline phosphatase in the rat liver for cytochemical study]

A perfusion method is described for the ultrastructural demonstration of alkaline phosphatase. The present approach is based on a short vascular perfusion of the rat liver with glutaraldehyde through the portal vein or the aorta, followed by a vascular perfusion incubation with a medium containing the enzyme substrates. From the vessels a good penetration of the medium into all tissues is achieved. It is demonstrated that a 3 minute glutaraldehyde perfusion provides a good morphological preservation of cells. For alkaline phosphatase, the reaction product was confined to the inner layer hepatocyte plasma membranes. No signs of enzyme diffusion were noted. The present approach seems to offer some advantages: it is simple and requires no extra equipment, penetration of the fixative and incubation enzyme medium is good, and freeze artifacts are avoided.