Measurement of NADPH-cytochrome P-450 reductase content in rat liver sections by quantitative immunohistochemistry...

We have a quantitative light microscopic immunohistochemical method using video image processing. First, an antigen (NADPH-cytochrome P-450 reductase) content in homogenates of livers of rats was measured by enzyme immunoassay. Then frozen sections from rat livers were incubated with the anti-NADPH-cytochrome P-450 reductase antibody under saturation conditions by the indirect immunoperoxidase method. Subsequently, relative staining intensities in small portions and those in wide areas in the sections were measured with a video image processor. Finally, the resulting relative values obtained from the small portions were converted into absolute NADPH-cytochrome P-450 reductase contents using the results of enzyme immunoassay and the average relative staining intensity obtained from the wide areas in the sections. The reductase content in sections from rat livers measured by the image processing method coincided with the content measured by the microphotometric method using a nitrocellulose model system. The present image processing method is applicable to measurement of contents of antigens that can not be immobilized in model systems.