Claudin 5

Representative paraffin blocks - defined as those with the largest amount of viable and anaplastic tumor - for each tumor were selected. Immunohistochemistry was performed on the tumor tissues and the neonatal ependyma and choroid plexus. The immunohistochemical reactions were performed on 3 μm sections obtained from the FFPE blocks. After the deparaffinization steps, the slides were treated in a microwave oven in Target Retrieval Solution (S1699 from DAKO, Carpenteria, CA, USA) for 30 min for heat-induced epitope retrieval. The immunohistochemical reactions were performed in an automated Ventana ES Immunostainer System (Ventana Medical Systems Inc., Tucson, AZ, USA) with the solutions and steps according to the manufacturer. The following antibodies and dilutions were used: claudin-1 (1:80, Zymed, #18–7362), claudin-2 (1:20, Zymed, #18–7363), claudin-5 (1:120, Zymed, #18–7364), claudin-7 (1:100, Zymed, #34–9100), E-cadherin (1:500, Dako #M3612), N-cadherin (1:300, Abcam #12,221), occludin (1:250, Invitrogen, Carlsbad, CA, USA ) and vimentin (1:300, Dako #M0725). The slides were counterstained with Mayer’s hematoxylin (Zymed, South San Fransisco, CA, USA). Positive controls and negative control tissues (with the omission of the primary antibodies) were included in every run.

RBMEC were grown on collagen coated Labtek chambered slides (Nunc). Cells were fixed with 4% para-formaldehyde (PAF) on ice for 10 min, and permeabilised for 5 min with 0.1% Triton X-100 in PBS. The cells were then blocked in 10% normal goat serum diluted in PBS for 1 h. The primary antibodies were applied for 1 h at RT (PECAM-1: mouse, 1:50, Serotec; ZO-1: rabbit, 2.5 µg/mL, Zymed; Occludin: mouse IgG1-k 10 µg/mL, Zymed; Claudin-3: rabbit, 10 µg/mL, Zymed; Claudin-5: mouse, 10 µg/mL, Zymed). After 3 washes with PBS, the corresponding fluorescence-conjugated secondary antibody (rabbit-IgG: goat, 10 µg/mL, Invitrogen; mouse-IgG: goat, 10 µg/mL, Invitrogen) was added to the cells and incubated for 1 h. After 3 final washes, the slides were mounted with a glass coverslip with Dako Fluorescent Mounting Medium (DakoCytomation) and viewed with a fluorescence microscope.