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. 2021 Jun 6;9:105. doi: 10.1186/s40478-021-01209-3

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — Immunostaining of phospho-NRF2 (S40) in the brain regions of PFF aSyn injected M83^+/+ mice. A Representative IHC images showing phospho-NRF2 (S40) immunostaining with distinct nuclear staining in brainstem regions (red arrows). Also, notice the predominantly cytoplasmic staining in cerebellar lobules (purkinje cells), motor cortex and corpus striatum-yellow arrows (10 × low magnification views and 40 × magnified views in the insets; scale bar = 200 µm; Aq. in the image showing PAG, cerebral aqueduct; 4v. in the image showing vestibular nuclei image, 4th ventricle). Bregma co-ordinates for the brain regions were determined, according to Paxinos and Franklin: (Bregma, − 3.63 mm) midbrain at the level of superior colliculi showing PAG, red nucleus and tegmentum; (Bregma, − 5.79) pontocerebellar junction showing cerebellar nuclei, cerebellar lobules (cb1-5), vestibular nuclei and pontine gigantocellualr nuclei (Gi); and (Bregma, 0.73 mm) forebrain showing motor cortex (M1 and M2) and corpus striatum. Also see Additional file 1: Fig. S5A (PBS injected cohort) and Additional file 1: Fig. S6B, C (additional high resolution data from the PFF and PBS cohorts). Primary antibody in A: p-NRF2 (S40)-PA5-67520. B Semi-quantitative analyses of p-NRF2 (pS40) immunopositive cells in the indicated brain regions of PBS or PFF injected M83^+/+ mice. Individual data points represent p-NRF2 (pS40) cell counts/mm²/region in each animal of the respective cohort, with PBS cohort being depicted as blank triangles and PFF as the black squares. (PAG periaqueductal grey matter; RN red nucleus; Gi pontine gigantocellualr nuclei; VN pontine vestibular nuclei; one-way ANOVA, ***p < 0.0001; n = 5/group)