Immunostaining Analysis of Axonal Transport and Neuronal Integrity

Immunostaining for amyloid precursor protein (APP) was used as a surrogate marker for impairment of axonal transport because it is known to accumulate when transport function in axons is blocked (46 (link)). Neuronal somatodendritic integrity was assessed by immunostaining for the cytoskeletal protein microtubule-associated protein-2 (MAP-2). Assessment of microglia morphology was performed with immunostaining for Iba-1. Immunohistochemistry was performed on 50 µm free-floating sections under moderate shaking. Before staining, the sections were incubated 30 min in 0.3% hydrogen peroxide to quench endogenous peroxidases. After three washing steps in 0.1 M phosphate buffer (pH 7.4), non-specific antibody binding sites were blocked with using 10% normal goat serum. Different free-floating sections were incubated overnight at 4 °C with anti-MAP-2 (1 : 500, monoclonal mouse-IgG; Millipore, MAB3418), anti-APP (1 : 500, monoclonal mouse-IgG; Millipore, MAB348), or anti-Iba-1 (1 : 500, polyclonal rabbit-IgG; Wako, 019-19741) in 5% normal goat serum. After several washes, sections were incubated for 2 h at room temperature with secondary antibodies. For APP and MAP-2 staining, biotinylated anti-mouse-IgG, 1 : 500, Vector, BA9200 was used, and the streptavidin/horseradish peroxidase detection was performed according to the manufacturer's recommendations. These sections were incubated with the substrate diaminobenzidine (DAB, D3939; Sigma-Aldrich Company, St. Louis, MO, United States) for 10 min at room temperature. For Iba-1 staining, Alexa Fluor 594 sary antibody (1:500, Invitrogen, #A-11037) was used. Immunofluorescent images were obtained with a Leica microscope (model DMi8 with THUNDER Imager 3D software).