Vector abc

Lumbar or sacral mouse or rat spinal cord sections were used for preembedding immunoperoxidase labeling of VGLUT3. Mouse tissue sections were initially incubated in 1% NaBH₄ in PBS for 30 min to quench free aldehyde groups. After permeabilization in 50% ethanol for 30 min and incubation in PBS with 1% BSA for 1 h, the sections were incubated in primary antibody solution (mouse anti-VGLUT3, 1:1000 in PBS with 1% BSA) at room temperature overnight or for 72 h. After rinsing, the sections were subsequently incubated in biotinylated goat anti-mouse secondary antibody (Vector Laboratories) in PBS with 1% BSA and in Vector ABC (Vector Laboratories) for 2–3 h each. Peroxidase activity was visualized by incubation in ImmPACT DAB (Vector Laboratories) for 30 s to 2 min. Sections thus labeled for VGLUT3 were rinsed briefly in PB (0.1 M; pH 7.4), incubated in 0.5–1% OsO₄ in PB for 10–30 min (depending on section thickness), dehydrated in graded series of ethanol and embedded in Durcupan (Electron Microscopy Sciences). Ultrathin sections of embedded tissue were counterstained using 2% uranyl acetate (15 min) or UranyLess (2 min; Electron Microscopy Sciences) followed by 0.4% lead citrate before examination in a JEOL 1230 electron microscope.

Immunohistochemistry for Aβ plaques was performed on 5 μm sagittal sections. The antibody used was 20G10 (Glaxo Smith Kline, Harlow, UK; 0.28 μg/mL) (Howlett et al., 2004 (link)) raised against the Aβ_35–42 fragment and selected for its C-terminal Aβ₄₂ specificity. Immunohistochemistry was completed with appropriate secondary biotinylated antibodies (Vector Laboratories, Peterborough, UK) diluted in 1:500 in secondary layer diluent (0.3% Triton-X-100 in 0.1 M PBS), followed by avidin-biotin complexation (Vector ABC, Vector Laboratories), and visualized using diaminobenzidine according to the manufacturer's data sheets (Vector Laboratories). The percentage of the total section area labeled by the Aβ antibody in representative sections from each mouse fed either the control or (−)-epicatechin diet was determined using Qwin software macros (Leica, V2.0) (Howlett et al., 2004 (link)).