Mouse anti ap 2α

Restriction endonucleases were purchased from New England Biolabs, Inc. (Ipswich, MA, USA). Rabbit anti-TFPI-2, goat anti-TFPI-2 and mouse anti-AP-2α antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, USA). Mouse anti-TFPI-2 antibody was purchased from R&D systems (Minneapolis, MN, USA). Mouse anti-MMP2 antibody was from Novus Biologicals (Littleton, CO, USA). Anti-β-actin, anti-tubulin-α and anti-lamin B antibodies were purchased from Bios Inc. (Tokyo, Japan). Goat anti-rabbit and goat anti-mouse antibodies were purchased from Sigma-Aldrich Chemical Co (St. Louis, MO, USA). Cy3-conjuagted donkey anti-rabbit, anti-mouse and anti-goat antibodies, FITC-conjugated donkey anti-goat and anti-rabbit antibodies were purchased from Jackson Immunoresearch Laboratories (West Grove, PA, USA).

Deparaffinized sections of P18 mice were incubated for 20 minutes in citrate buffer (10mM, pH 6.0) at 98°C in a steam bath for antigen retrieval, washed with PBS and permeabilized by PBS with 0.1% Tween 20, blocked with 10% BSA and incubated with primary antibody at 4°C overnight. The following primary antibodies were used: mouse anti-Ap2α (Santa-Cruz, 12726X, 1:3000), mouse anti-Calbindin-D-28K (Sigma-Aldrich, clone CB-955, C9848, 1:2500), mouse anti-Lim1/2 (DSHB, clone 4F2-c, 1:300), rabbit cone arrestin (Millipore, AB15282, 1:1000), rabbit anti-Pax6 AF1 (1:3000), rabbit Sox9 (Millipore, 1:500), rabbit anti-Tbr2 (Abcam, ab23345, 1:500), sheep anti-Chx10 (ThermoFisher Scientific, PA1-12565, 1:500). Next day, sections were washed with PBS, incubated with fluorescent secondary antibody Alexa Fluor 488 or 594 (Life Technologies, 1:500) for 1 hour at room temperature, washed with PBS, counterstained with DAPI (1 μg/ml) and mounted in mowiol (4–88; Sigma).