Goat anti rabbit fab fragment

For double immunoelectron microscopy, sections were cryoprotected and freeze-thawed. After blocking in 20% NGS in 50 mM TBS, sections were incubated with primary antibodies for GFAP (rabbit, polyclonal, DAKO) and Reelin (mouse-monoclonal, clone G10, Millipore, Billerica, MA, USA) in 50 mM TBS containing 3% NGS (Vector Laboratories, Burlingame, CA) for 24h at 4°C. After washes in TBS, the sections were incubated with biotinylated goat anti-mouse IgG antibody (1:100; Vector Laboratories) and goat anti-rabbit (Fab fragment, 1:100) coupled to 1.4 nm gold (Nanoprobes, Stony Brook, NY). Subsequently, sections were processed for silver enhancement of the gold particles with an HQ Silver kit (Nanoprobes) and incubated with avidin-biotin peroxidase complex (ABC kit; Vector Laboratories) that was visualized with 3,3′-diaminobenzidine tetrahydrochloride (0.05%) as a chromogen and 0.01% H₂O₂ as substrate. Sections were then treated with 1% osmium tetroxide and uranyl acetate, dehydrated and flat-embedded in epoxy resin (Durcupan ACM Fluka; Sigma-Aldrich). Ultrathin sections were cut at 60–70 nm on an ultramicrotome (Reichert Ultracut E; Leica), and viewed on a Philips CM100 electron microscope. Images were taken with a CCD camera (Orius SC600; GATAN) and analyzed using GATAN imaging software.