Abt45

Mice were transcardially perfused, as previously reported [25 (link),26 (link)]. Their brain and colon tissues were post-fixed, cytoprotected, frozen, sectioned, and washed [19 (link),26 (link)]. The washed section was blocked with serum, incubated with primary antibodies for BDNF (1:200, Santa Cruz Biotechnology, #SC-65513, canonical nerve growth factor), NeuN (1:200, Millipore, #MAB377, a neuronal nuclear antigen), NF-κB (1:100, Cell Signaling Technology, #3033S), Iba1 (1:200, Thermo Fisher Scientific, #PA5-27436, a microglial protein), LPS (1:200, Abcam, #ab35654, a Gram-negative bacterial endotoxin), MUC2 (1:500, Abcam, #ab272692), claudin-1 (1:200, Santa Cruz, #G0119, an intestinal tight junction protein), claudin-5 (1:200, Millipore, ABT45, a brain tight junction protein), and/or CD11c (1:200, Abcam, #ab11029, an immune cell protein) overnight, washed, and incubated with the secondary antibodies conjugated with Alexa Fluor 594 (1:200, Invitrogen) or Alexa Fluor 488 (1:200, Invitrogen) for 2 h. The section was also stained with DAPI and photographed by a confocal laser microscope.

Cells in the NVU were individually scraped down and lysed on ice for 10 min. After centrifugation (13,000g, 4°C), the resulting supernatant was saved as the cytoplasmic extract sample and the nuclear pellet was prepared for a nuclear extract sample. The samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (P0012A, Beyotime, China) and then transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h and incubated overnight at 4°C with the following antibodies: rabbit polyclonal antibody against GAP-43 (1 : 1000, 8945S, CST, China), rabbit polyclonal against AQP-4 (1 : 1000, ab31721, Abcam, China), rabbit polyclonal antibody against Claudin-5 (1 : 1000, ABT45, Millipore, China), and mouse polyclonal antibody against Tubulin (1 : 200, sc-5286, Santa Cruz, China). Membranes were incubated with a secondary goat anti-rabbit/mouse antibody (1 : 3,000, Service, China) for 1 h at 37°C. Immunoreactive bands were observed using the ECL detection system (Bio-Rad, Beijing, China).

After 48 h of MCAO/R, ischaemic hemisphere in brains was collected and disrupted using RIPA buffer (high) (Shandong Sparkjade Biotechnology Co., Ltd.). Quantitative immunoblotting was performed as previously described (Hu et al. 2021 (link)). Anti-β-tubulin (Millipore, Burlington, MA) was used as an internal loading control. The images of blots were captured using an Azure C300 with secondary antibodies. The images were captured using Image Lab, and the signal intensities were normalized to loading controls. The antibodies and their concentrations are as following: (antibody, company, catalog number, dilution): Anti-Claudin-5, Millipore, ABT45, 1:500; and Anti-Aquaporin-4 (AQP4), Millipore, ABN411, 1:500.