A CellProfiler Pipeline for Quantification of p-SNCA in Mouse Striatal Cholinergic and Medium Spiny Neurons

This protocol describes an automated image analysis workflow using CellProfiler (1) to quantify Ser129 phosphorylated α-synuclein (pSNCA) within Cholinergic and Medium Spiny Neurons (MSNs) in the striatum of Synuclein G51D mutant mice (2). Brain sections are immunostained for Ser129 phospho-α-synuclein, DARPP-32 to identify MSNs, choline acetyltransferase (ChAT) to label cholinergic neurons, and DAPI to identify nuclei. Confocal Z-stacks are processed into consecutive three-slice maximum-intensity projections (MIPs) using a custom FIJI/ImageJ macro prior to segmentation and analysis. Quantitative outputs include integrated pSNCA intensity and neuronal mask area, which are normalised using a custom R script to yield cell type specific pSNCA burden per unit area. This workflow enables cell type resolved quantification of α-synuclein pathology from fluorescence imaging datasets.