α-synuclein pathology in the brainstem and the SNpc region were visualized by staining with anti-pS129 antibody (1:1000; Abcam). The semi-quantitative grading of p-α-Syn pathology of the SNpc was quantified as previously described [16 (link)] with minor modification. The samples were graded using a 0-3 semi-quantitative density scale.
Microglia and astrocyte were stained with anti-Iba-1 (1:1000; Wako) or anti-GFAP (1:2000; Dako), antibodies followed by incubation with biotin-conjugated anti-rabbit antibody and ABC reagents (Vector Laboratories). Then, sections were developed using SigmaFast DAB Peroxidase Substrate (Sigma-Aldrich, St. Louis, MO, USA). The number of microglia and densities of astrocyte in the SNpc region were measured using ImageJ software. The GlcCer-positive signals were stained with anti-GlcCer antibody (1:500, Glycobiotech), followed by incubation with CY3-conjugated anti-donkey secondary antibody. The fluorescent images were acquired through a Zeiss confocal microscope (LSM 710, Zeiss Confocal).
Microglia and astrocyte were stained with anti-Iba-1 (1:1000; Wako) or anti-GFAP (1:2000; Dako), antibodies followed by incubation with biotin-conjugated anti-rabbit antibody and ABC reagents (Vector Laboratories). Then, sections were developed using SigmaFast DAB Peroxidase Substrate (Sigma-Aldrich, St. Louis, MO, USA). The number of microglia and densities of astrocyte in the SNpc region were measured using ImageJ software. The GlcCer-positive signals were stained with anti-GlcCer antibody (1:500, Glycobiotech), followed by incubation with CY3-conjugated anti-donkey secondary antibody. The fluorescent images were acquired through a Zeiss confocal microscope (LSM 710, Zeiss Confocal).
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