Immunostaining of phospho-alpha synuclein (S129) in brain regions of PFF aSyn injected M83^+\+ mice and assessment of hindlimb clasping behaviour. (A–B) Representative images showing p-aSyn (S129) IHC in indicated brain regions at dpi 21 (A) and dpi 45 (B) (dpi, days post-injection; 10× magnified views, scale bar = 200 µm). Yellow arrows in the insets point to select instances of p-aSyn (S129) positive oligodendroglial cells in the vicinity of neurons. Also see Supplementary Fig. 2A–B (representative images from PBS injected- dpi 45 and PFF aSyn injected- dpi 14 cohorts) and Supplementary Fig. 3A (5× low magnification images annotated with neuroanatomical landmarks, PFF aSyn injected- dpi 45). Primary antibody in A–B: p-aSyn (S129)- 11A5. (C) Semi-quantitative analyses of p-aSyn (S129) immunopositive cells in the indicated brain regions at dpi 21 and dpi 45 in PFF injected M83^+/+ mice. Individual data point represents p-aSyn (S129) cell counts/mm²/region in each animal of the respective cohort, with dpi 21 being depicted as blank triangles and dpi 45 as the orange squares. (MdV, medullary reticular nuclei; Gi, pontine gigantocellualr nuclei; VN, pontine vestibular nuclei; PAG, periaqueductal grey matter; RN, red nucleus, DCN- crb, deep cerebellar nuclei; M1+M2, primary and secondary motor cortex; One-way ANOVA, Kruskal–Wallis test- p.0002, n = 5/group; dpi, days post-injection). (D) Hindlimb clasping assessment in PBS and PFF aSyn injected cohorts, with individual data points representing clasping score of each animal in the respective cohort. Notice that PBS injected mice do not exhibit hindlimb clasping (blank circles, score = 0). The PFF injected mice start show mild clasping around dpi 21–28 and later (blue squares; 4 animals at dpi 21–28 and 6 animals at dpi 45). Two-way ANOVA (alpha 0.05- P < 0.0001; n = 8/group).