Anti aqp4

Mice were perfused with cold PBS followed by 4% paraformaldehyde 24 h after focal cerebral ischemia. Immediately thereafter, brains were harvested and further fixed for 24 h in 4% paraformaldehyde, following which they were cryoprotected in 30% sucrose for 72 h at 4°C. Each brain was frozen in optical cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA) and stored at −80°C until analysis. The frozen brains were sectioned (thickness: 20 μm) using a CM 3050 cryostat (Leica Microsystems, Wetzlar, Germany). Brain sections were immunostained with anti-ZO-1 (1 : 100), antioccludin (1 : 100, Invitrogen Corporation, Carlsbad, CA), anti-CD-31(1 : 100, BD Bioscience), and anti-AQP4 (1 : 100, Millipore) overnight at 4°C, following which they were incubated with Alexa 488 or Alexa 594-conjugated secondary antibodies (1 : 500, Life Technologies) for 2 h in total darkness. DAPI (molecular probe) was used for nuclei staining. Fluorescence images were captured using a Zeiss LSM 700 laser scanning confocal device (Carl Zeiss, Jena, Germany) and Slide Scanner Axio Scan.Z1 (Carl Zeiss, Jena, Germany). The images are quantified using Metamorph Microscopy Automation and Image Analysis Software (Molecular Devices, USA) and i-Solution software (Image & Microscope Technology, Vancouver, Canada).

The following primary antibodies were used: rabbit polyclonal anti-AQP4 (Sigma, Saint Louis, Missouri, USA) diluted to 1:2000 for immunoblot analysis and 1:1000 for immunofluorescence; rabbit polyclonal anti-α1-Syntrophin (Sigma, Saint Louis, Missouri, USA) at 1:1000 for immunofluorescence experiments; mouse monoclonal anti-human Dystrophin (Monosan, PB Uden, The Netherlands) used at 1:500 for immunofluorescence; mouse monoclonal anti-CD31 (Dako, Santa Clara, California, USA) diluited to 1:100 for immunofluorescence experiments; Custom rabbit polycolonal anti mouse AQP4ex generated against the peptide DSTEGRRDSLDLASC within the mouse AQP4 carboxy-extension (GenScript) and diluted 1:5000 for immunoblotting and 1:4000 for immunofluorescence. For immunofluorescence, AlexaFluor 488 anti-rabbit, AlexaFluor 488 anti-human, AlexaFluor 488 anti-mouse and AlexaFluor 594 anti-mouse were used (all from Life Technologies, Thermo Fisher Scientific, Carlsbad, California, USA) at a dilution of 1:1000; for immunoblotting, anti-rabbit IgG-HRP (Bio-Rad, California, USA) was used at 1:3000.

Anti-agrin labelling was performed using a polyclonal rabbit antibody received as a kind gift from Professor Markus A. Ruegg, University of Basel. The primary antibody solution was diluted 1:500 for immunogold labelling and 1:1000 for immunoperoxidase staining. Anti-laminin labelling was done using a rabbit polyclonal antibody (Sigma-Aldrich L9393) diluted 1:100 for immunoperoxidase and immunogold labelling.
Visualisation of anti-agrin and anti-laminin staining for electron microscopy was done using a goat anti-rabbit antibody conjugated with 15-nm colloidal gold particles (Abcam) and diluted 1:20. For immunoperoxidase staining, a biotinylated donkey anti-rabbit (Pierce) secondary antibody diluted 1:100 was used for both anti-agrin and anti-laminin experiments.
The primary antibodies used for immunofluorescence were anti-β-dystroglycan (1:100 dilution; beta-dystroglycan (H-242) antibody, Santa Cruz Biotechnology; Cat# sc-28535; RRID:AB_782259) and anti-AQP4 (1:400 dilution; Sigma Aldrich; Cat# A5971; RRID:AB_258270). Cy3 donkey anti-rabbit (Jackson ImmunoRe-search Labs; Cat#: 711-165-152; RRID:AB_2307443) was used as secondary antibody in a 1:500 dilution. Vessels were stained using DyLight^® 649 conjugated tomato lectin (LEL, TL; Vector labs; Cat#: DL-1178).

Small blocks of the cortex were subjected to freeze substitution and infiltration in Lowicryl HM20 resin (Polysciences, Warrington, PA, Cat 15924) (Schwarz & Humbel, 1989). Using a Reichert ultramicrotome (Wien, Austria) sections of 80 nm thickness were cut, mounted on nickel grids and further processed for immunogold cytochemistry. Specifically, the sections were incubated in the following solutions: (1) 50 mM glycine in Tris buffer (5 mM) containing 0.01% Triton X‐100 and 50 mM NaCl (TBST; 10 min); (2) 2% human serum albumin (HSA) in TBST (10 min); (3) primary antibody (anti‐AQP4 from Sigma, 25 μg mL⁻¹) diluted in the solution used in the preceding step (overnight); (4) same solution as in step 2 (10 min × 2); and (5) gold‐conjugated IgG (GAR15 nm for human sections and GAR10 nm for mouse sections, both obtained from Abcam), diluted 1:20 in TBST containing 2% HSA and polyethylene glycol (0.5 mg mL⁻¹, 1 h). Sections were then counterstained.

The cells were seeded evenly in 24-well plates with 7 × 10⁴ cells per well. After treatments, the cells were washed with cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 minutes, washed with PBS, and infiltrated with a 3% BSA solution containing 0.3% Triton X-100 (#T824275; Mecklin, Shanghai, China) for 1 hour. Cells were then co-incubated with the following antibodies: anti-GFAP (#130300; 1 : 300; Invitrogen), anti-AQP4 (#ABN411; 1 : 300; Sigma-Aldrich, USA), anti-phosphor-p38 (#4511s; 1 : 200; CST), or anti-phosphor-NF-κB p65 (#3033s; 1 : 200; CST) overnight at 4°C in a humidity chamber. The next day, the cells were washed and incubated for 1 hour at room temperature with goat anti-rat IgG/Cy3 (#bs-0923G-Cy3; 1 : 400; Bioss) to label GFAP and Alexa Fluor 488-conjugated goat anti-rabbit IgG (#A11034; 1 : 400; Invitrogen) to label AQP4, phosphor-p38, and phosphor-NF-κB p65. Finally, the coverslips were rinsed in PBS and mounted in a 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting medium (#ZLI-9557; ZSGB-BIO, Beijing, China). Images were acquired using an LSM780 inverted confocal microscope (Carl Zeiss). All images were captured under the same conditions within a given experiment, and 3–5 independent experiments were carried out. The average fluorescence intensity was analyzed using ZEN software (version 2.3).