Fig. 2

α-synucleinopathy is found within neuronal somata and some of it colocalizes with the ubiquitin marker of protein aggregates. a Two month-old mice were unilaterally infused with α-synuclein fibrils (5 μg) or an equal volume of phosphate-buffered saline (PBS) into the olfactory bulb and adjoining anterior olfactory nucleus (OB/AON). Fibrils were sonicated for 1 h in a waterbath prior to infusion. Three months later, sagittal brain sections were collected and stained with antibodies against pSer129 and the neuronal nuclear marker NeuN. Hoechst-labeled nuclei are shown in blue. Arrows point to some examples of the many triple-labeled cells in the AON. b Confocal microscopy showing the perinuclear localization of pSer129⁺ structures in the AON. Arrows: examples of Hoechst⁺/NeuN⁺/pSer129⁺ profiles. Arrowhead: Hoechst⁺/NeuN⁻/pSer129⁺ profile. For three-dimensional movies of pSer129⁺ and NeuN⁺ cells, see Additional file 2. c Sagittal sections through the OB/AON were stained with the Thioflavin amyloid stain. Nuclei were labeled blue with Hoechst. An additional sagittal level can be viewed in Additional file 1: Figure S4. d Colocalization of ubiquitin, pSer129, and NeuN in five different brain regions in mice that were sacrificed 3 months after fibril infusions in the OB/AON (1 h waterbath sonication). Some, but not all pSer129⁺ structures were ubiquitin⁺. The pSer129 and ubiquitin photos were captured from fibril and PBS-treated animals at the same exposures and intensity scaling. To view the results of the second ubiquitin antibody, please see Additional file 1: Figure S5. e Mice were infused in the OB/AON with 5 μg α-synuclein fibrils (1 h waterbath-sonicated) or PBS and perfused 1.5 h later. Brain sections were stained with rabbit antibodies against phosphorylated or total α-synuclein (red). The needle track is still apparent in the caudal OB at this early timepoint (white arrows) and reveals variability in the precise site of infusion, which would result in differences in inclusion counts in those afferent neurons terminating in the OB/AON in highly topographical manners. Images from PBS and fibril groups were captured at the same intensity scaling and exposure times. Differences in background staining are not the result of differential image processing. For a high-resolution version of this figure, please download the image at the link at the end of this article (Additional file 3) or email the corresponding author at leakr@duq.edu for access to the files