Occludin

For immunohistochemistry, cold saline of 10 ml and paraformaldehyde of 4% in 0.1 M PBS were used to anesthetize mice by perfusing through the heart. Then we harvested the brain quickly, and post-fixed in PFA of 4% for 48 h, and immersed in sucrose solution of 30%. They were storaged at 4 °C prior to be sectioned. Frozen sections were then cut by a thickness of 10 μm. Sections floating freely were washed by phosphate-buffered saline and blocked with Triton X-100 of 0.3% and normal blocking serum in PBS (10%) for 1 h at room temperature. Then they were stained overnight at 4 °C using the Claudin-5 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Occludin (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD13(1:50; Abcam, Cambridge, United Kingdom), Laminin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and β-amyloid (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibody. The next day, PBS was used to wash sections and sections were incubated for 2 h with Cy3 conjugated anti-mouse IgG and Cy3 conjugated anti-rabbit IgG (1:100; Jackson immune Research Laboratories). Cells were stained by Occludin, Claudin-5, CD13, Laminin (red) or DAPI (blue). They were analyzed through an upright microscope (Ni-E, Nikon Corporation, Tokyo, Japan).

Antibodies used for Western blotting and immunofluorescence assays were as follows: hnRNPM (OriGene), hnRNPF (Santa Cruz Biotechnology, Inc.), hnRNPH1/H2 (Santa Cruz Biotechnology, Inc.), p-Smad3 (Cell Signaling), Smad3 (Cell Signaling), CD44 (R&D Systems and Santa Cruz Biotechnology, Inc.), E-cadherin (Cell Signaling Technology), γ-catenin (Cell Signaling Technology), occludin (Santa Cruz Biotechnology, Inc.), N-cadherin (BD), vimentin (NEOMarkers), β-actin (Sigma-Aldrich), and GAPDH (Millipore). The ESRP1 antibody was a gift from Dr. R. Carstens (University of Pennsylvania).