Mab5364

Antibodies for CTIP2 (ab18465, 1:500), CUX1 (ab54583, 1:500), PE-conjugated anti EGFR (ab231, 1:50), LGALS1 (ab25138, 1:1,000), Lin28 (ab46020, 1:1,000), PHH3 (ab5176, 1:250), PLZF (ab104854, 1:100), POU3F2 (ab94977, 1:1,000), SATB2 (ab51502, 1:50), SOX1 (1:1,000), SOX2 (ab79351, 1:500), TBR1 (ab31940, 1:200), TBR2 (ab23345, 1:200) were from Abcam. Antibodies for BrdU (347580, 50 μl per test), KI67 (556003, 1:1,000), phycoerythrin-conjugated (PE) SSEA-3 (560237, 20 μl per test), PE-conjugated F11R (552556; 20 μl per test), Alexa Fluor 647-conjugated TRA-1-60-647 (560850, 5 μl per test), Alexa Fluor 647-conjugated TUJ1 (560340, 1:500) were from BD Biosciences. Antibodies for DCX (AB2253, 1:5,000), O4 (MAB345, 1:25), RELN (MAB5364, 1:200), Tyrosine Hydroxylase (TH, AB152, 1:500) were purchased from Millipore. Antibodies for FABP7 (51010-1-AP, 1:100), S100B (15146-1-AP, 1:100) were from ProteinTech. Antibodies for AP2α (3B5 concentrated, 1:100) and PAX6 (supernatant, 1:16) were from DSHB. Antibody for NESTIN (MO15012, 1:500) was from Neuromics. Antibody for GFAP (Z0334, 1:2,000) was from DAKO. Antibody for β-3-Tubulin (PRB-435P, 1:1,000) was from Covance. Antibody for GLAST (ACSA-1; 130-095-822, 1:10) was from Miltenyi Biotec. Antibody for OTX2 (AF1979; 1:40) was from R&D. Antibody for FOXA2 (SC-6554; 1:100) was from Santa Cruz Biotechnology.

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 was performed as previously described [21 (link),22 (link)]. The primary antibodies and dilutions were as follows: anti-Calretinin (Millipore, AB5054, 1:500, Billerica, MA, USA); anti-Ctip2 (Abcam, ab18465, 1:2000, Cambridge, MA, USA); anti-Foxg1 (Abcam, ab18259, 1:1000, Cambridge, MA, USA); anti-GFP (Abcam, ab13970, 1:1000, Cambridge, MA, USA); anti-Lhx2 (Abcam, ab184337, 1:500, Cambridge, MA, USA); anti-P73 (Abcam, ab40658, 1:500, Cambridge, MA, USA); anti-Prox1 (Millipore, AB5475, 1:1000, Billerica, MA, USA); and anti-Reelin (Millipore, MAB5364, 1:1000, Billerica, MA, USA). The secondary antibodies used were Alexa Fluro 488 donkey anti-chicken (Jackson Lab, 703-545-155, 1:500, West Grove, PA, USA), Alexa Fluor 546 donkey anti-rabbit (Life, A10040, 1:500, Gaithersburg, MD, USA), Alexa Fluro 647 donkey anti-rabbit (Life, A31573, 1:500, Gaithersburg, MD, USA), Alexa Fluor 546 donkey anti-rat (Life, A10040, 1:500, Gaithersburg, MD, USA), CF 568 donkey anti-rat (Sigma-Aldrich, SAB4600077, 1:500, St. Louis, MO, USA), CF 633 donkey anti-rat (Sigma-Aldrich, SAB4600133, 1:500, St. Louis, MO, USA), and Alexa Fluro 647 donkey anti-mouse (Invitrogen, A21236, 1:500, Carlsbad, CA, USA).