Rabbit anti human cldn5

Markers of BBB integrity were assessed by measuring serum concentrations of S100β, occludin (OCLN), claudin-5 (CLN5), and zonula occludens 1 (Zo-1). To estimate OCLN concentrations, rabbit and mouse anti-human OCLN antibodies (Zymed Labolatories Inc, South San Francisco, CA, USA) were used as capture and detection antibodies, respectively. CLDN5 concentrations were estimated with an in-house enzyme-linked immunosorbent assay (ELISA), using mouse anti-human CLDN5 (Zymed Labolatories Inc.) and rabbit anti-human CLDN5 (Abcam Inc., Cambridge, UK) antibodies for capture and detection, respectively. Zo-1 levels were analysed with rabbit anti-human Zo-1 and mouse anti-human Zo-1 (Invitrogen, Waltham, MA, USA) antibodies for capture and detection, respectively. Goat anti-mouse IgG (H+L)−HRPO was used as the secondary antibody for the OCLN and Zo-1 ELISAs, and goat anti-rabbit IgG (H+L)−HRPO (Invitrogen) was used for the CLDN5 ELISA.
S100β levels were determined using commercially available ELISA kits according to the manufacturer’s instructions (BioVendor Laboratory Medicine Inc., Brno, Czech Republic; and R&D Systems Inc., Minneapolis, MN, USA).

After 48 h of transfection, cells were collected, and 50 mL RIPA lysis buffer (Thermo) with 1% protease inhibitor was added to every 5 × 10⁵ cells on ice for 30 min. After centrifugation at 12,000 rpm for 10 min at 4°C, the supernatant was collected to determine protein concentration using a BCA protein assay. The supernatant was boiled for 5–10 min and then stored at‐20°C. The extracted protein was subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for 1 h, then transferred to a polyvinylidene fluoride membrane for 20 min, and sealed in milk solution for 2 h. With the addition of rabbit anti‐human CLDN5 (Abcam) and mouse anti‐human, the membrane was maintained overnight at 4°C. Subsequently, HRP‐labeled goat anti‐rabbit IgG antibody and HRP‐labeled rabbit anti‐mouse IgG antibody were added, and samples were incubated at 37°C. Chemiluminescence visualization was performed in the dark according to the instructions of the electrochemiluminescence color kit (Thermo).