Anti claudin 5 mouse monoclonal antibody

The eye tissues were fixed in 10% PBS-buffered formaldehyde, and embedded in paraffin subsequently. Serial 4 μm sections were prepared from paraffin blocks. After deparaffinization, endogenous peroxidase was quenched with 3% H₂O₂ in H₂O for 5 min. Sections were incubated in 5% adult bovine serum (ABS) in PBS for one hour to minimize nonspecific adsorption. Then they were incubated overnight with anti-TNFSF15 rabbit polyclonal antibody (1:750 in 5% BSA) (Abcam, Cambridge, MA, USA), anti-GFP rabbit polyclonal antibody (1:1000 in 5% BSA) (Abcam, Cambridge, MA, USA), anti-VEGF rabbit polyclonal antibody (1:250 in 5% BSA) (Abcam, Cambridge, MA, USA), anti-claudin-5 mouse monoclonal antibody (1:100 in 5% BSA) (Invitrogen, Camarillo, CA, USA), anti-occludin rabbit polyclonal antibody (1:400 in 5% BSA) (Abcam, Cambridge, MA, USA), or anti-ZO-1 rabbit polyclonal antibody (1:400 in 5% BSA) (Invitrogen). Specific labelling was detected with biotin-conjugated goat anti-mouse IgG or goat anti-rabbit IgG and avidin-biotin peroxidase complex. Positive staining appeared brown.

Evaluation of the expression and localization of the TJ protein claudin‐5 after Aβ peptide treatment in CMEC cells was evaluated by immunocytochemistry (ICC) as previously described (Fossati et al., 2010). Briefly, cells were plated on collagen‐coated glass chamber slides (Thermo Fisher Scientific) and were grown in EBM‐2 0.25% FBS +bFGF 1:2000 to facilitate the development of tight junctions. After cells were confluent for 5 days, cells were treated for 24 h with 10 μM of Aβ42 oligomers, Aβ42 fibrils, or freshly solubilized Aβ42, washed with PBS, and subsequently fixed for with 4% paraformaldehyde at room temperature. Claudin‐5 was visualized by incubation with anti‐claudin‐5 mouse monoclonal antibody (Invitrogen), followed by an incubation with Alexa Fluor 488‐conjugated anti‐mouse IgG (Invitrogen) and nuclei visualized by counterstaining with To‐pro (Invitrogen). Images were then acquired using Nikon Eclipse Ti inverted fluorescence microscope with deconvolution. Specificity of the antibody detection was assessed by omission of the primary antibody during the ICC procedure.

The rabbit anti-c-fos polyclonal antibody, rabbit anti-caspase-3 polyclonal antibody, rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) polyclonal antibody, and rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody were purchased from Abcam (Abcam, USA). The monoclonal mouse anti-claudin-5 antibody was purchased from Invitrogen (Invitrogen, USA). The monoclonal mouse anti-matrix metalloproteinase-2 (MMP-2), monoclonal mouse anti-MMP-9, and monoclonal rabbit anti-occludin antibodies were purchased from Abcam (Abcam, USA). RIPA buffer and the BCA kit were purchased from Beyotime (Shanghai, China). The mouse IL-6 ELISA kit and the IL-1β ELISA kit were obtained from R&D Systems (Minneapolis, MN, USA).