Polyclonal rabbit anti zo 1

Total proteins were extracted from epididymal tissues using Tissue Protein Extraction Reagent (Thermo Scientific) containing 1 mM of PMSF (Roche). The protein concentrations were determined by BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein samples were separated by SDS/PAGE (10% (w/v) gel) and transferred on to PVDF membranes (Millipore, Billerica, MA, U.S.A.). The transferred proteins were incubated with polyclonal rabbit anti-ZO-1 (1:500, Santa Cruz Biotechnology), polyclonal rabbit anti-vimentin (1:2000, CST, Danvers, MA, U.S.A.), and polyclonal rabbit anti-GAPDH (1:10000, Abcam) primary antibodies at 4°C overnight, followed by incubation with the horseradish peroxidase conjugated anti-rabbit (1:10000, KPL, Milford, MA, U.S.A.) secondary antibody for 1 h at 37°C. The reaction was developed with the ECL kit (Pierce Chemical, Dallas, TX, U.S.A.) and photographed.

All primary and secondary antibodies were dissolved in PBS (1:200) prior to labeling. The PBECs, astrocytes and pericytes were fixed in 4% paraformaldehyde and blocked in PBS supplemented with 0.2% Triton-X-100 and 3% bovine serum albumin for 1 hour. The PBECs were stained with polyclonal rabbit anti-claudin-5 (Sigma-Aldrich, cat. no. SAB4502981, lot 310145) and polyclonal rabbit anti-ZO-1 (Invitrogen, cat. no. 617300, lot 1087989A). Mixed glial cells were stained with rabbit anti-glial fibrillary acidic protein (GFAP)(DAKO, DK, cat. no. Z0334, lot 20003791) and Texas Red labelled Lycopersicon Esculentum (Tomato) Lectin (Vector Labs, Peterborough, United Kingdom, cat. no. TL1176, lot W0812). Pericytes were stained with monoclonal mouse anti-α-smooth muscle actin (α-SMA) (Sigma-Aldrich, cat. no. A5228, lot 091M4832), polyclonal rabbit anti-ZO-1 and rabbit anti-platelet-derived growth factor receptor-beta (PDGFR-β) (Santa Cruz, cat.no.Sc-432, lot K1113). For detection, the cells were subsequently stained with goat anti-rabbit Alexa 488 or goat anti-mouse Alexa 585 (Invitrogen) as the secondary antibodies. All cells were counterstained with DAPI. The Millicell membranes were cut out of the inserts and mounted on glass slides in fluorescent mounting media (Dako, Denmark) and cover slips were placed upon the membranes.