Abstract
<p>Quantification of neuronal populations in thick, free-floating tissue sections is inherently time-consuming and subject to observer variability. To address these limitations, this protocol employs semi-automated DAPI-based nuclear segmentation and immunofluorescence (IF) classification in QuPath (v0.7.0) to provide a reproducible, standardized, and time-efficient approach for estimating the abundance of dopaminergic neuronal subtypes in mouse midbrain tissue. This workflow combines nuclear morphology-based cell detection with marker-specific classification to identify and quantify neuronal populations expressing tyrosine hydroxylase (TH), aldehyde dehydrogenase 1 family member A1 (Aldh1a1), calbindin 1, and their co-expression patterns. Although the method does not provide unbiased stereological counts and remains dependent on the accuracy of tissue staining, image segmentation, and classifier performance, it enables reliable and reproducible comparisons of relative neuronal abundance between experimental groups. By analyzing anatomically matched sections within a defined bregma range, the protocol provides a practical approach for assessing regional and subtype-specific changes in dopaminergic neurons within the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). The workflow also includes procedures for identifying and quantifying double-labelled neuronal populations, facilitating the characterization of molecularly distinct dopaminergic neuron subtypes in experimental mouse models.</p>