Claudin 11

Cells plated in 4-well chamber slides coated with Fibronectin (5 µg/ml, BD Biosciences) were exposed to DZ-50 treatment (5 µM) for 12 hrs. Cells were then fixed in 100% (v/v) methanol, and after blocking at 4°C (5% NGS, 0.3% Triton X), were exposed to the primary antibody (4°C, overnight). The following specific antibodies were used: ILK-1 (Cell Signaling Technology), ZO-1 (Invitrogen), Claudin-11 (Santa Cruz Biotechnology), Snail (Cell Signaling Technology), Talin (Millipore). Cells were then incubated with fluorochrome-conjugated secondary antibody (Invitrogen) (2 hrs, room temperature) and subjected to confocal microscopy using an Olympus FV1000 Confocal Microscope v1.21. and software version FV10-ASW 3.1.

Histological sections (5 μm) were deparaffinized in xylene and rehydrated in 100, 95, 80, and 70% ethanol. Antigen retrieval was performed with pH 6.0 buffer citrate and endogenous peroxidase blocking with 0.3 % H₂O₂ in PBS. Sections were incubated overnight in 1% bovine serum albumin in PBS at room temperature. Primary antibodies (claudin-11 and N-cadherin, both 1/200 dilution: Santa Cruz, Dallas, USA; occludin and TGF-β3, 1/50 and 1/100 dilution, respectively: Abcam, Cambridge, UK) were incubated overnight at 4°C in a moisturized chamber and the day after peroxidase-conjugated secondary antibody (1/50 dilution; Pierce anti-rabbit, anti-goat, and anti-mouse, Cambridge, UK) was added and reaction visualized with 3,3′-Diaminobenzidine (Sigma–Aldrich, Milan, Italy). Counterstaining was performed with haematoxylin alone. Negative control slices were tested using PBS instead of primary antibody.

Whole brain homogenates (including the hippocampus) were mixed with SDS sample buffer and heated to 90 °C for 5 min. Proteins were separated by SDS-PAGE in a Mini-Protean electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA) and electroblotted overnight onto polyvinylidene difluoride membranes. Membranes were probed with primary antibodies against Myelin-associated glycoprotein (MAG) (Mouse anti-mouse/rat/human, 1:1000, Abcam), Myelin-associated oligodendrocyte basic protein (MOBP) (Rabbit anti-mouse/human, 1:500, Abcam), 2′,3′-Cyclic nucleotide 3′-phosphodiesterase (CNP) (Mouse anti-mouse, 1:2000, Sigma), myelin oligodendrocyte glycoprotein (MOG) (Rabbit anti-mouse, 1:500, Sigma), Myelin basic protein (MBP) (Rat anti-mouse, 1:50, Abcam), Claudin11 (Rabbit anti-mouse/rat/human, 1:100, Santa Cruz). The blots were washed and incubated for 1 h at room temperature (RT) with AP-conjugated secondary antibody (1:1000; Dako, Glostrup, Denmark). Immunodetection was performed using the ECF immunoblotting detection system for AP-conjugated secondary antibody (GE Healthcare, Diegem, Belgium). Blots were scanned with the FLA-5000 (Fuji Photo Film Corp.). Relative amounts of immunoreactivity were quantified using Quantity One 1-D software (Bio-Rad). To correct for input differences, Coomassie protein staining (the upper or lower half of the same gel for ECF) was used.