Ultrascan 4000sp ccd

To evaluate the ultrastructural characteristics of DAI in the micro pig thalamus while verifying microglia process contacts on these axonal swellings, a subset of tissue was immunolabeled with either rabbit anti β-APP (1:700; Cat.# 51–2700, Life Technologies, Carlsbad, CA, USA) or rabbit anti Iba-1 (1:1000; Cat.# 019–19741, Wako, Osaka, Japan), followed by incubation with biotinylated goat anti-rabbit IgG (1:1000; Cat.# BA-1000, Vector Laboratories, Burlingame, CA, USA) secondary antibody. The reaction product was visualized with 0.05 % diaminobenzidine/0.01 % hydrogen peroxide/0.3 % imidazole in 0.1 M phosphate buffer and the tissue was prepared for EM analysis. In this approach, tissue sections were osmicated, dehydrated, and embedded in epoxy resin on plastic slides. After resin curing, the slides were studied with routine light microscopy to identify the precise thalamic areas for excision. Once identified, these sites were removed, mounted on plastic studs, and 70-nm sections were cut serially and mounted on Formvar-coated slotted grids. The grids were stained in 5 % uranyl acetate in 50 % methanol and 0.5 % lead citrate. Ultrastructural qualitative analysis was performed using a JEOL JEM 1230 transmission electron microscope (JEOL-USA, Peabody, MA, USA) equipped with Ultrascan 4000SP CCD and Orius SC1000 CCD cameras (Gatan, Pleasanton, CA, USA).

To verify microglia process contacts on axonal swellings 1 day following cFPI in the micro pig, tissue was assessed for microglial process contacts as described previously (25 (link)). Briefly, a subset of tissue was immunolabeled with rabbit anti- Iba-1 (1:1,000; Cat.# 019-19741, Wako Osaka Japan) followed by incubation with biotinylated goat anti-rabbit IgG (1:1,000; Cat# BA-1000, Vector Laboratories, Burlingame, CA, USA) secondary antibody. The reaction product was visualized with 0.05% diaminobenzidine/0.01% hydrogen peroxide/0.3% imidazole in 0.1 M phosphate buffer and the tissue was prepared for EM analysis. In this approach, tissue sections were osmicated, dehydrated, and embedded in epoxy resin on plastic slides. After resin curing, the slides were studied with routine light microscopy to identify the precise thalamic areas for excision. Once identified, these sites were removed, mounted on plastic studs, and 70-nm sections were cut serially and mounted on Formvar-coated slotted grids. The grids were stained in 5% uranyl acetate in 50% methanol and 0.5% lead citrate. Ultrastructural qualitative analysis was performed using a JEOL JEM 1230 transmission electron microscope (JEOL-USA, Peabody, MA, USA) equipped with Ultrascan 4000SP CCD and Orius SC1000 CCD cameras (Gatan, Pleasanton, CA, USA).