Anti fading mounting medium

For the time course analysis of Reelin expression following KA application, OHSC were treated with KA (10 μM) for 45 min followed by incubation in fresh medium. Slices were fixed at 0 (immediately after KA treatment), 1, 2, 3, 4, 6, and 8 h post-KA with 4% paraformaldehyde (PFA, Roth) in 0.1 M phosphate buffer (PB; pH 7.4; 4 h at RT) and subsequently rinsed several times in PB. Immunolabeling for Reelin was performed using a free-floating protocol (Orcinha et al., 2016 (link)). After pre-treatment (0.25% Triton X-100, 10% normal serum in PB, 2 h at RT), slices were incubated (0.1% Triton X-100, 1% normal serum in PB, 24 h at RT) with a mouse monoclonal anti-Reelin antibody (G10, 1:1.000, Millipore, Cat# MAB5364, RRID: AB_2179313). Antibody binding was visualized by incubation with a goat anti-mouse Cy5-conjugated secondary antibody (1:400, RRID: AB_2338714, Jackson ImmunoResearch Laboratories) in the dark (6 h at RT) and counterstained with DAPI (4′,6-diamidino-2-phenylindole; 1:10.000, Roche). Whole slices were dried on glass slides, coverslipped with anti-fading mounting medium (DAKO, Sigma Aldrich, United States) and stored in the dark at 4°C.

Immunofluorescence staining refers to the methods used by Zi-YunYi et al. (24 (link)), In brief, the oocytes in each group were fixed in 4% paraformaldehyde in a PBS with 0.5% Triton X-100 for 1 h at 4°C, followed by blocking in 3% BSA for 1 h at 37°C. Thereafter the oocytes were incubated with mouse monoclonal anti-γ-tubulin antibody (4D11, MA1850, invitrogen, 1:30) overnight at 4°C. After two washes (10 min each) in a washing buffer (0.1% PVA in PBS), the oocytes were labeled with Goat Anti-Mouse IgG H&L (DyLight^® 594, ab96881, Abcam, 1:30) for 1 h at 37°C. After two washes, the oocytes were stained with monoclonal anti-β-tubulin-FITC (F2043-2ML, Sigma, 1:30) for 1 h at 37°C, then co-stained with DAPI for 10 min at room temperature, followed by two more washes. Finally, the oocytes were mounted on glass slides with an antifading mounting medium (Sigma), and visualized with a confocal laser-scanning microscope (Nikon Ti2, Japan).