The study of brain pathology by fluorescence microscopy finds in the autofluorescence of the tissue an additional difficulty for the recognition of markers of interest. In particular, in the immunofluorescence study of brains from Alzheimer's disease (AD) patients, several approaches have been attempted to eliminate or mask the presence of autofluorescent aggregates. In the present work, we propose a method to characterize by fluorescent microscopy senile plaques discriminating them from autofluorescent aggregates, such as lipofuscin granules.This work describes four protocols carried out in human brain tissue of patients with AD, covering adequate tissue preparation, immunofluorescence acquisition, and data analysis: 1. Tissues processing of frozen samples for optimal epitope conservation. 2. Analysis of the fluorescence emission spectrum of the tissue by performing a confocal microscopy λ-scan. 3. Analysis of fluorescence emission of both intact and formic acid-treated tissues in four channels corresponding to the emission in blue, green, near red, and far-red regions. 4. Analysis a specific immunostaining of amyloid beta in senile plaques, using fluorescent-labeled antibodies by using specific emission channels to avoid detection of tissue autofluorescence.
Keywords: Alzheimer’s disease; Autofluorescence; Brain tissue; Confocal microscopy; Immunofluorescence; Lipofuscin.