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 microscopy (CLSM) images of immunofluorescence and lipofuscin in human brain cortical areas is described. Indirect immunofluorescence was used to show the distribution and density of a synaptic vesicle protein - synaptophysin (p38). Dual channel CLSM was used for imaging immunofluorescence and autofluorescence of lipofuscin granules. Special software was developed for quantitative analysis of the CLSM images. The method has been tested in studies of frontal, temporal, motor, visual, and entorhinal cortices in normal and pathological human brains (Rett syndrome and autism). Using this technique the intensity and amount of p38 immunoreactivity was quantified after subtraction of lipofuscin fluorescence. In normal cortex no statistically significant differences were found for p38 antigen between layers II, III and V. The highest concentration of p38 immunoreactivity was found in the frontal cortex. In Rett syndrome and autistic patients, the p38 immunofluorescence in the investigated cortex areas appeared reduced compared with normal. The software is also suitable for quantitative analysis of double-labelled immunofluorescent specimens in animals without lipofuscin.