QUANTITATIVE-ANALYSIS OF IMMUNOFLUORESCENCE AND LIPOFUSCIN DISTRIBUTION IN HUMAN CORTICAL AREAS BY DUAL-CHANNEL CONFOCAL LASER-SCANNING MICROSCOPY

A method for quantitative analysis of confocal laser scanning microsco py (CLSM) images of immunofluorescence and lipofuscin in human brain c ortical areas is described. Indirect immunofluorescence was used to sh ow the distribution and density of a synaptic vesicle protein - synapt ophysin (p38). Dual channel CLSM was used for imaging immunofluorescen ce and autofluorescence of lipofuscin granules. Special software was d eveloped for quantitative analysis of the CLSM images. The method has been tested in studies of frontal, temporal, motor, visual, and entorh inal cortices in normal and pathological human brains (Rett syndrome a nd autism). Using this technique the intensity and amount of p38 immun oreactivity was quantified after subtraction of lipofuscin fluorescenc e. In normal cortex no statistically significant differences were foun d for p38 antigen between layers II, III and V. The highest concentrat ion of p38 immunoreactivity was found in the frontal cortex. In Rett s yndrome and autistic patients, the p38 immunofluorescence in the inves tigated cortex areas appeared reduced compared with normal. The softwa re is also suitable for quantitative analysis of double-labelled immun ofluorescent specimens in animals without lipofuscin.