Mouse anti ocln

Two sections per brain were selected from the ventral hippocampus approximately 300 μm apart. Immunofluorescence to detect the expression of claudin-5 (CLDN5), occludin (OCLN) and endothelial nitric oxide synthase (eNOS) was performed on lectin-injected tissues. Brain sections were initially washed and permeabilized with PBS and Triton^™ (0.1%) and blocking was performed in 10% goat serum, 0.2% bovine serum albumin (BSA) and 0.1% Tween^®. Brain sections were incubated overnight at 4°C in 3% goat serum, 0.1% BSA and 0.1% Tween with the primary antibodies against CLDN5 (Rabbit anti-CLDN5 1:200), OCLN (Mouse anti-OCLN, 1:250) and eNOS (Rabbit anti-eNOS 1:250), all from Invitrogen^™ (Carlsbad, CA). The next day, tissues were incubated for 1 h at room temperature with the following secondary antibodies: Goat anti-Mouse 647 (1:500) and Goat anti-Rabbit 594 (1:400), both from Abcam ^® (Cambridge, MA) and Goat anti-Rabbit 647 (1:400; Thermo Fisher Scientific^™ Inc., Waltham, MA), before counterstaining with DAPI. Tissues were then slide mounted and imaged at 40× using a Nikon Eclipse Ti-E Confocal (Tokyo, Japan) or Olympus^® FV3000 Confocal (Tokyo, Japan) at 40×.