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Phenotypic changes in vesicular compartments are an early pathological hallmark of many peripheral and central diseases. For example, accurate assessment of early endosome pathology is crucial to the study of Down syndrome (DS) and Alzheimer's disease (AD), as well as other neurological disorders with endosomal-lysosomal pathology. We describe a method for quantification of immunolabeled early endosomes within transmitter-identified basal forebrain cholinergic neurons (BFCNs) using 3-dimensional (3D) reconstructed confocal z-stacks emplo