A quantitative approach to efficiency and sensitivity: enzyme histochemistry, immunofluorescence and PAP-technique in...

Aminopeptidase was demonstrated and its concentration measured in the bile canaliculi of frozen pig liver sections. Sections were stained for enzymic activity by conventional histochemical means, by using purified rabbit anti-aminopeptidase followed by fluorescent labelled anti-rabbit-immunoglobulin and rabbit peroxidase anti-peroxidase complex. Absorption and fluorescence measurements by microscopic photometry revealed that under the conditions used the minimal amount of reaction product detectable by the PAP-technique is 3 to 10 times smaller than that of fluorescent stain by immunofluorescence technique, i. e. the PAP-technique is 3 to 10 times more efficient and relative to the amount of detectable anti-aminopeptidase approximately 3 times more sensitive. For the given enzyme concentration in the tissue these two techniques were as sensitive as conventional histochemistry. Saturation of the sites of an antigen within the tissue with antibody is essential for quantitative determination of antigens.