Figure 3.

Immunostaining and Western Blot Analyses of Different Segments from Diseased EUS-α-Syn PFFs TgM83^+/− Mice and Age-Matched EUS-PBS TgM83^+/− Mice

(A–D) Immunohistochemical results from EUS-PBS TgM83^+/− mice (A1–D1) and diseased EUS-α-Syn PFFs TgM83^+/− mice (A2-D2) for calbindin-D28k in the periaqueductal gray (A) and Barrington nucleus (B) and MAP-2 in the intermediolateral columns of L2 (C) and pelvic ganglia (D). (A3–D3) Quantification of the number (A3, B3) and optical density (C3, D3) of neurons shown in (A1–D1 and A2–D2).

(E–H) Double immunofluorescence analysis of the L2 level of the spinal cord (E), substantia nigra (F), and pons (G, H) from EUS-PBS TgM83^+/− mice (F1–H1) and diseased EUS-α-Syn PFFs TgM83^+/− mice (E, F2–H2) for pα-Syn (red, E, H) with ubiquitin (Ub) (green, E1), Iba-1 (green, E2), and glial fibrillary acidic protein (GFAP) (green, H); Ub (red, F) with tyrosine hydroxylase (TH) (green, F); and neurofilament (red, G) with MBP (green, G). (E3, F3) Quantification of colocalization of Ub with pα-Syn (E3) and TH (F3) shown in (E1, F1, F2). (G3, H3) Quantification of optical density of MBP (G3) and GFAP (H3) shown in (G1, G2, H1, H2). Co-immunolabeling is represented by signal in yellow. Cell nuclei were counterstained with Hoechst 33258 (blue). EUS-α-Syn PFFs TgM83^+/− mice, n = 20; EUS-PBS TgM83^+/−, mice n = 10. Scale bars: 200 μm in (F1, F2), 50 μm in (A1–D1, A2–D2), and 40 μm in (E1, E2, G1, G2, H1, H2).

(I–N) Representative immunoblots (I, K, M) and quantification (J, L, N) of pα-Syn in the sarkosyl-soluble and insoluble fractions of spinal cord (I, J), pons (K, L), and PAG (M, N). Blots were probed for GAPDH as a loading control (bottom). Molecular weight markers of migrated protein standards are expressed in kDa. n = 3 animals per group.

Data are the means ± SD. Statistical significance was analyzed by using the Student's t test and Mann-Whitney test, ∗∗∗∗p < 0.0001, ∗∗p < 0.01, ∗p < 0.05; n.s., non-significant.