Ab53765

Immuno-histochemistry was done on 6 μm thick cryo-sections of CP from healthy donors and from donors diagnosed with AD (Braak stage 6). Cryostate sections were fixed for 10 min in 4 % paraformaldehyde in 0.1 M phosphate buffer. For staining we employed standard procedures. CLDN5 was detected with Cy3 coupled secondary antibodies after labeling with anti-Claudin 5 antibody ab53765 (Abcam). Double staining for Transthyretin (TTR) and the tight junction protein 1 (ZO-1) was done using the Anti-TTR/Transthyretin Antibody (clone 10E1) IHC-plus™ LS-B2864, from LSBio, visualized with a Cy3 coupled secondary antibody (goat anti mouse, Jackson ImmunoResearch), and the ZO-1 antibody (Zymed 61-7300), visualized with Alexa 647 coupled secondary antibody (donkey anti rabbit, Jackson ImmunoResearch). Slides were photographed on a Leica SP8 confocal microscope.

Tissue sections were deparaffinised in xylene (Sigma-Aldrich) and rehydrated to distilled water in decreasing concentrations of ethanol (Commercial Alcohols, Inc). Heat-induced antigen-retrieval (HIAR) was carried out in Tris-EDTA buffer (10 mM Tris, 1 mM EDTA Solution, 0.05% Tween 20, pH 9.0; Sigma-Aldrich) for 30 min at 90°C. Slides were blocked for 3 hrs at room temperature in 5% (w/v) skim milk (Bio-Rad) in PBSA and then incubated overnight at 4°C with a 1 : 250 dilution of rabbit anti-CLDN4 (ab53156, Abcam, Cambridge, MA, USA) or 1 : 100 dilution of rabbit anti-CLDN5 (ab53765, Abcam) in incubation buffer (1% BSA, 1% Donkey Serum, and 0.5% Triton X-100 in PBS; Sigma-Aldrich). Slides were then washed three time in PBS and incubated in a 1 : 500 dilution of FITC-labeled goat anti-rabbit IgG (4030-02, Southern Biosystems, Birmingham, AL, USA) for anti-CLDN4 or PE-labeled goat anti-rabbit IgG (ab97070; Abcam) in incubation buffer at 4°C for 4 hours. Slides were again washed three times in PBS before the cover slip was added with Prolong Gold antifade with DAPI (Life Technologies). Intestinal villi were imaged using an Axiovert 200 M with a 63X neoFluor objective (Zeiss, Oberkochen, Germany) under oil immersion. Finally, DAPI and FITC/PE images were background subtracted and merged using ImageJ software [51 (link)].