Ab18465

The differentiated human iPSCs were fixed on glass coverslips (VWR, Denmark) in 4% PFA for 15 min at RT and stored at 4°C until use. The cells were washed in PBS and permeabilized in 0.1% Triton X-100 in PBS for 1 h, washed in PBS and antigen retrieval was performed by immersing cells into boiling one x citrate buffer 3 times for 5 min each. Cells were treated with blocking buffer (5% NDS in PBS) for 1 h at RT. Cells were incubated overnight at four°C in primary antibodies targeted against BCL11B (1:750, Abcam, ab18465), Reelin (1:50, Santa-Cruz, sc-25346), SATB2 (1:800, Abcam ab34735), Nestin (1:500, Millipore, Abd69) and MAP2ab (1:200, Sigma, M1406) diluted in blocking buffer. The cells were washed 3 × 5 min in PBS and incubated with secondary antibodies conjugated with Alexa fluorophores (1:200, Invitrogen, A10036, A21208, and A21448) diluted in blocking buffer for 1 h at RT. The cells were then washed three times for 5 min in PBS and counterstained with Hoechst 33342 (1 μg/ml in PBS) for 10 min. The cells were washed in PBS and mounted onto glass microscope slides using buffered glycerol mounting media 90% glycerol (Sigma-Aldrich), 20 mM Tris pH 8.0, 0.5% N-propyl gallate (Sigma-Aldrich).

Immunohistochemical analyses were performed as previously described (Colombo et al., 2006 (link)). The primary antibodies utilized were the following: anti-CTIP2 (Abcam ab18465), anti-CUX1 (santa Cruz Biotechnology sc-13024), anti-SATB2 (Abcam, ab51502), anti-PAX6 (Covance #PRB-278P), anti GFAP (chicken, 1:1,000, Abcam, ab4674), anti-DCX (rabbit, 1:1,000, Abcam, ab18723), antiKI67 (rabbit, 1:500, immunological sciences, mab-90948), anti-NEUN (rabbit, 1:500, Abcam, ab104225).
Secondary antibodies: 488-mouse (donkey, 1:2,000, Molecular Probes, A21202), 488-rabbit (donkey, 1:2,000, Molecular Probes, A21026), 594-mouse (donkey, 1:2,000, Molecular Probes, A10036), 594-rabbit (donkey, 1:2,000, Molecular Probes, A21207). DAPI (4′,6′-diamidino-2-phenylindole) was used as nuclear counterstaining.

Mice (5 WTs and 5 Hets for POU3F2 staining, 7 WTs and 6 Hets for BCL11B staining, sex balanced, at the age of P60) were deeply anesthetized and transcardially perfused with 4% paraformaldehyde in PBS. Whole brains were weighed and serially sectioned in the coronal plane at 75 μm using a vibratome and immunolabeled for either BCL11B (a marker for cortical layers V/VI) or POU3F2 (a marker for cortical layers II-IV). For each antibody, a set consisting of every eighth section was isolated and slide mounted. After drying overnight, antigen retrieval was performed by immersing in citrate buffer (pH 6.0) and pressure cooking for 10 min. The slides were then quenched in 3% hydrogen peroxide in absolute methanol for 10 min, immersed for 1 h in a blocking solution (2% bovine serum albumin, 0.2% dry milk, 0.8% Triton X-100 in PBS), and incubated overnight with a 1:500 dilution of either BCL11B (Abcam ab18465) or POU3F2 (Santa Cruz sc-393324). The next morning, BCL11B or POU3F2 incubated sections were reacted with appropriate biotinylated secondary antibody for 1 h (Sigma-Aldrich B7139; 1:200 or Vector Labs BA-9200; 1:200, respectively). The sections were then reacted with an avidin-biotin conjugate (ABC kit) for 1 h and visualized using the chromogen VIP (Vectastatin Elite ABC kit and Vector VIP kits; Vector Labs).