Reelin

As previously published [53 (link)], cell lysates or tissue pieces were prepared by adding protease and phosphatase inhibitor cocktail in RIPA buffer and centrifuging for 10 min at 12,000 rpm to remove debris. From the lysates, protein concentrations were determined by the Lowry protein assay (500-0113, 500-0114, 500-0115; Bio-Rad (Hercules, CA, USA)). Equal amounts of protein were loaded into each lane of a 4–12% Tris gel (BioRad) and subjected to electrophoresis. After blotting, nitrocellulose membranes (BioRad) were blocked for 1 h (milk powder 5% in TBS/tween 0.1–0.2%) and incubated with primary antibodies (ICAM-1, R&D Systems, AF796; E-Selectin, Santa Cruz, sc-137054; G10 anti-Reelin, made in house; Reelin, Millipore, MAB5366; GAPDH, Sigma-Aldrich, G8795). The binding of secondary HRP-antibodies was visualized by ECL or ECL plus chemiluminescent (Amersham). After densitometric analyses with ImageJ, optical density values were expressed as arbitrary units and normalized for protein loading, as described in the figure legends.

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.

Prior to proceeding with the IHC/IF protocol, a standard process of deparaffinizing sections was performed in xylol and alcohol series. Antigen retrieval was performed by boiling sections in citrate buffer (pH 6,0). After three washes in PBS, blocking solution containing 1–3% BSA and 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MI, USA) in PBS was applied on the sections for 1–2 h. The blocking solution was replaced with primary antibodies diluted in a blocking solution and incubated overnight at 4 °C. The following antibodies were used: NeuN (Abcam, Cambridge, UK), MAP2 (Sigma-Aldrich, St. Louis, MI, USA), MAP2 (Merck Millipore, Burlington, MA, USA), CUX2 (Abnova, Taipei, Taiwan), CUX2 (Abcam, Cambridge, UK), Reelin (Millipore), DCX (Merck Millipore, Burlington, MA, USA), FOXP1 (Abcam, Cambridge, UK), Neuroserpin (Abcam, Cambridge, UK), TLE4 (Santa Cruz Biotechnology, Dallas, TX, USA), Nurr1 (R&D systems, Minneapolis, MN, USA). After incubation, sections were washed in PBS, and appropriate secondary antibodies (Alexa Fluor, Thermo Fisher Scientific, Waltham, MA, USA) were applied for 2 h at RT. Following three washes in PBS, TrueBlack quencher (Biotium, Fremont, CA, USA) was applied on the sections. Finally, the sections were covered using a mounting medium with DAPI (Vectorlabs, Burlingame, CA, USA). The IHC protocol was implemented as previously described [32 (link)].