Anti claudin 5 antibody

Following dewaxing, rehydration and antigen retrieval, the 4-μm sections of the brain tissue were incubated with hydrogen peroxide to block endogenous peroxidase, then blocked with 5% bovine serum albumin (BSA; Sigma) for 1 h. The sections were incubated with anti-claudin-5 antibody (1:50, Invitrogen) at 4 °C overnight and then with secondary antibody (1:100; Abcam) for 30 min at 37 °C. Peroxidase activity was visualized with 3-diaminobenzidine (DAB). Then, the slides were stained with hematoxylin, dehydrated with a gradient alcohol, cleared with xylene, and cover slipped.

Freshly isolated microvessels were spread onto glass microscope slides and heat-fixed for 10 min at 95 °C, followed by 4% paraformaldehyde (PFA) for 15 min at room temperature. Samples were then washed three times with phosphate buffered saline (PBS) and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Nonspecific binding was blocked with 10% normal goat serum (Abcam) in PBS for an hour and the microvessels were incubated with anti-ZO-1 antibody (Invitrogen) or anti-claudin-5 antibody (Invitrogen) overnight at 4  °C, followed by incubating with goat anti-rabbit antibody conjugated with Alexa Flour 488 (Invitrogen) for 2 hours at room temperature. Then, the microvessels were incubated with anti-occludin antibody conjugated with Alexa Flour 594 (Invitrogen) overnight at 4 °C. After three washings with PBS, slides were mounted with Vectashield HardSet Mounting Medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector laboratory, CA). Images were acquired using a confocal microscope (Olympus Fluoview V5; Olympus America, Melville, NY). Microvessels sized between 4 and 8 μm in diameter were selected for analyses and at least 7 different microvessel images per each mouse sample were used for the quantification. The total area of analyzed microvessels from a mouse sample was between 12,000~26,000 μm².

Immunostaining for claudin-5 and ZO-1 was performed using a procedure described previously [22 (link)]. Briefly, brains were removed from mice who underwent transcardial saline perfusion after 24 h of reperfusion. The brain tissues were fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich, St. Louis, MO, USA) for 24 h at 4 °C and then transferred to 30% sucrose in phosphate buffered saline (PBS) for 48 h at 4 °C. The tissues were embedded in Tissue-Tek optimal cutting temperature (OCT) compound (Sakura Finetek Inc, Torrance, CA, USA), frozen at −70 °C for 30 min, and sectioned at 20 μm using a frozen section machine (Meyer Instruments, Houston, TX, USA) at −33 °C; the sections were mounted on poly-D-lysine-coated glass slides and soaked in 3% bovine serum albumin (BSA) blocking solution at 25 °C. Sections were incubated with anti-ZO-1 antibody (1:50, Invitrogen, Carlsbad, CA, USA) or anti-claudin-5 antibody (1:100, Invitrogen) overnight and then with secondary antibody labeled with Alexa 568 (1:500; Invitrogen). Finally, the sections were incubated with fluorescein isothiocyanate (FITC)-labeled Ulex europaeus agglutinin-1 (UEA-1; Sigma Aldrich). All samples were observed under a Nikon C2 confocal microscope (Nikon, Tokyo, Japan).

Western blot analysis was performed by modifying a procedure described previously [24 (link)]. The cells were lysed and centrifuged at 14,000 rpm for 15 min and the supernatant was collected to obtain the whole-cell lysate. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reacted with anti-ZO-1 antibody (1:1000, Invitrogen) or anti-claudin-5 antibody (1:1000, Invitrogen) overnight. All samples were analyzed using the LAS 4000 mini (Fuji Photo Film, Tokyo, Japan). All cells were pretreated with 30 μg/mL HSP 30 min before hypoxia exposure.

Western blot analysis was performed as described [21 (link)]. Tissues were harvested from control, S-W, and MSC groups (12 hours before surgical wound) at 6 hours after surgical wound. Anti-Claudin-5 antibody (Invitrogen, USA), anti-Occludin antibody (Abcam, USA), anti-ZO-1 antibody (Abcam, USA), anti-matrix metalloproteinase-9 (MMP-9) antibody (Abcam, USA), anti-synaptophysin antibody (Abcam, USA), and anti-endothelial nitric oxide (eNOS) antibody (Abcam, USA) were performed in the analysis. The ratio of the levels of targeted proteins to the level of β-actin was performed to analyze the trend of those proteins in the brain tissues.

To detect the effect of virus infection on tight junction proteins in endothelial cells, cells were lysed in RIPA buffer (Beyotime). Proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes which were blocked with a blocking buffer (3% bovine serum albumin (BSA) in TBS with 0.05% Tween 20 (TBST)) and incubated with primary antibodies in the TBST in 4℃ overnight. Herein, the anti-ZO-1 antibody (Invitrogen), the anti-Occludin antibody (Invitrogen), the anti-Claudin 5 antibody (Invitrogen), the anti-vascular endothelial (VE)-Cadherin antibody (Abcam), and the anti-beta-Actin antibody (Senta Cruz) were utilized, respectively. After being washed three times with TBST, the membrane was probed with an HRP-conjugated secondary antibody and was developed with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher) and imaged on an Image Quant TM LAS 4000.

Immortalized hBMECs and iPSC-derived BMECs were infected as described above for 5 h at an MOI of 10. Then, cells were washed three times in sterile PBS, and cell lysates were taken using radioimmunoprecipitation assay (RIPA) buffer plus protease inhibitor cocktail (catalog number 78443; Thermo Scientific). Proteins were quantified using a bicinchoninic acid (BCA) assay (catalog number 23227; Thermo Scientific), and equal amounts were loaded onto protein gels and transferred to nitrocellulose membranes. Anti-COX IV antibody was used as a protein loading control (catalog number 4850T; Cell Signaling Technologies), and anti-claudin-5 antibody (catalog number 35-2500; Thermo Scientific) to visualize claudin-5. Horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Laboratory) and a BioRad ChemiDoc XRS+ instrument were used to image the blots.