Cyclic Multiplex Immunofluorescence Workflow for Mouse Models of Parkinson’s Disease v1

High-plex immunofluorescence (IF) is a powerful technique that has become popular among the scientific community in the recent years. Its appeal lies in enabling multi-target measurements on the same tissue area of interest with intact spatial relationships and lower sample burden, outperforming single- or low-plex immunohistochemistry approaches. Compared with conventional immunohistochemistry, multiplex IF delivers higher information density, robust co-expression mapping at cellular and subcellular resolution, quantitative comparability across cycles, and automation-ready workflows that increase throughput and reproducibility. Here, we present a step-by-step protocol for cyclic multiplex IF on mouse midbrain tissue. We describe procedures for tissue preparation, epitope retrieval, custom antibody conjugation, and automated cyclic IF using the MACSima‱ Platform (Miltenyi Biotec ‱). We also provide guidance on how to deal with metadata and data management, as well as basic image quality control. This protocol is optimized for studies of neurodegenerative disease and is readily applicable to mouse models of Parkinson’s disease pathology with an aim to resolve complex cellular microenvironments and biomarker patterns in situ.