Anti aqp4

Anti‐AQP4 was purchased from Santa Cruz Biotechnology (goat polyclonal, sc‐9888, Finnell ST 75220 Dallas, Texas) and diluted 1:100 for ELISA, 1:300 for immunofluorescence and 1:500 for immunoblotting and immunoprecipitation experiments 35, 36. The secondary antibodies used for Western blot were donkey anti‐goat (Santa Cruz Biotechnology, sc‐2020, Finnell ST 75220 Dallas, Texas) diluted 1:5000. The secondary antibody used for immunofluorescence was Alexa Fluor 488 anti‐goat (Molecular Probes, A‐11055 Eugene, Oregon, USA) diluted 1:1000.

Fixed brain hemispheres were cut into 40 µm sagittal sections with a vibratome (Leica). Sections were incubated with primary antibodies and were incubated overnight at 4°C, including anti-Aβ MOAB-2 (6C3) (Millipore) at 1:1000; and anti-GFAP (Invitrogen) at 1:500, anti-Iba1 (Wako) at 1:500; anti-synaptophysin (Santa Cruz) at 1:200, anti-pan-laminin (Sigma) at 1:300; anti-fibrinogen (Dako) at 1:2000; anti-AQP4 (Santa Cruz) at 1:100 or 1:200; anti-occludin (Invitrogen) at 1:100 in 2% normal horse serum in Tris-buffered saline (TBS)-Triton 0.02%. The following day, sections were incubated with appropriate secondary antibodies (1:400; Vector), followed by enhancement with avidin-biotin complex (ABC; Vector) and 3,3′-diaminobenzidine (DAB; Sigma). Images were acquired with a Leica DM2500 microscope connected to a camera (Q-Imaging Micropublisher 3.3 RTV) using Q-capture Pro software. For immunofluorescence, fluorescent secondary antibodies (1:400 Alexa Fluor^®, Invitrogen) were used. In some cases, staining with 647-labelled tomato lectin (Vector Labs) was carried out simultaneously with secondary antibody incubation. For Thioflavin S staining, sections were incubated with 1% Thioflavin S (Sigma) for 8 min before being washed with ethanol and H₂O.

The rats were sacrificed by decapitation at 2 and 8 weeks P.I. (n = 3 per time point per group). The whole brain cortex and a 10-mm lumbar SC segment from each rat were harvested for Western blot analysis. Proteins were separated by 12% SDS-PAGE electrophoresis and were blotted onto a polyvinylidene difluoride membrane. The membrane was subsequently incubated with rabbit polyclonal anti-GFAP (1:200; Thermo Fisher Scientific, Waltham, MA, USA), anti-AKT (1:500; R&D Systems, Minneapolis, MN, USA), anti-Tie2 (1:500; R&D Systems, Minneapolis, MN, USA), anti-AQP4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-AKT and pAKT (1:500; R&D Systems, Minneapolis, MN, USA), anti-Tie2 (1:2000; R&D Systems, Minneapolis, MN, USA), anti-αvβ3 and α5β1 integrins (1:1000; Neuromics, MN, USA), anti-caspase 3 (1:500; Cayman Chemical, Ann Arbor, MI, USA), and mouse anti-MBP (1:1000; Abcam, Cambridge, MA, USA) primary antibodies for 12 h at room temperature. To normalize protein bands to a gel loading control, the membranes were re-probed with rabbit anti-β-actin antibody (1:5,000; Abcam, MA, USA). The membranes were then incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5,000; Santa Cruz, CA, USA) and the blots were detected using enhanced chemiluminescence reagent. The incubation step with the primary antibody was omitted for the negative control.

Nitrocellulose membranes were blocked overnight at 4 °C with 2% bovine serum albumin (BSA) in Tris-buffered saline (TBS; in mM 10 Tris, 150 NaCl, pH 7.5, and 0.05% Tween 20®) and then incubated overnight at 4 °C in blocking solution containing the following antibodies: anti-Kir 4.1, anti-AQP-4, anti-COX1, anti-COX2 (diluted 1:1000), and anti-GS (diluted 1:10,000) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-actin (1:2000) (Sigma). Subsequently, the membranes were incubated for 1 h at room temperature in a solution containing horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (diluted 1:10,000), HRP anti-mouse IgG (diluted 1:10,000) (GEHealthcare, Sao Paulo, Brazil), or HRP anti-goat diluted 1:10000 (Sigma). A chemiluminescence signal was detected by luminol substrate reaction (ECL Western Blotting System, GEHealthcare®). Immunoblots were quantified by membrane scanning in an Image4000, GE Healthcare®, and optical densities of proteins studied were determined by ImageJ software (Packard Instrument Company) and the protein/actin ratio calculated.

Eight rats were deeply anesthetized by an intraperitoneal injection of pentobarbital sodium (25 mg/kg body weight, Nembutal, Kyoritsu Pharmaceutical Co., Ltd., Tokyo, Japan), and perfused through the heart with saline followed by 4% paraformaldehyde (PFA) in a 0.1 M PBS, pH 7.4. The retinas were carefully removed and post-fixed in 4% PFA in PBS overnight. After washing with PBS, the retinal tissues were immersed in 30% sucrose for 2–3 days at 4 °C and then embedded in OCT (optimal cutting temparature) compound (Sakura Finetechnical, Tokyo, Japan). Then, 4 μm frozen sections were cut with a cryostat (CM3050S, Leica, Wetzlar, Germany). After blocking with 1% normal goat serum plus 1% BSA (bovine serum albumin) and 0.1% Triton X-100 in PBS, the retinal sections were incubated overnight at 4 °C with the following primary antibodies: mouse monoclonal anti-VEGF (1:500, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-GFAP (1:500, Merck Millipore, Billerica, MA, USA), and anti-AQP4 (1:500, Santa Cruz). These sections were incubated for 2 h at 37 °C in Alexa 594 or Alexa 488-conjugated to the appropriate secondary antibodies (1:1000; Invitrogen, Carlsbad, CA, USA). The processed sections were photographed with a fluorescence microscope (BZ-X700, Keyence, Osaka, Japan).

Non-NMO, control-IgG-treated NMO, and anti-RGMa mAb-treated NMO rats were transcardially perfused using 4% PFA on day 21. Immunohistochemistry was performed using primary antibodies that marked cell type, including anti-AQP4 (1:50, Santa Cruz), anti-CD45 (1:20, BD) as a pan-leukocyte marker, anti- glial fibrillary acidic protein (GFAP; 1:100; Sigma-Aldrich) as a marker for astrocytes, anti-Iba1 (1:1000, Wako) as a marker for microglia and macrophages, anti- myelin basic protein (MBP; 1:500; Dako), and anti-SMI-312 (1:1000; Covance). Images were acquired using a FV-1200 laser-scanning confocal microscope or a BX51 fluorescence microscope (Olympus). The signal intensity of AQP4 and GFAP double-positive area was quantified by setting the threshold with Image J software (NIH). The number of preserved axons in the dorsal columns of the spinal cord were counted using Image J software (NIH).

Astrocytes were transferred to gelatin-coated microscope slides by cytospin (300 × g, 10 min) and fixed with 4% paraformaldehyde for 20 min at RT. Fixed cells were washed extensively with PBS and blocked with 10% rabbit normal blocking serum (Santa Cruz Biotechnology) supplemented with 0.3% Triton™ X-100 (Sigma-Aldrich) for 45 min at RT. Next, cells were washed and double-stained for IgG1 with AQP4, C5b-9 with phalloidin, and GFAP with C5b-9. Anti-IgG1/FITC (Dako Cytomation), anti-AQP4 (Santa Cruz Biotechnology), anti-C5b-9 (Dako Cytomation), phalloidin (Invitrogen), anti-GFAP (Santa Cruz Biotechnology), and rat IgG2b (Invitrogen), as negative isotype control were used. Secondary fluorescent antibodies [chicken pAb to rabbit Texas Red (TR) (Santa Cruz Biotechnology), goat pAbs to mouse FITC (Abcam), or goat pAbs to mouse TR (Thermo Fisher Scientific)] were added. The membrane localization of C5b-9, IgG1, and GFAP was analyzed by confocal microscopy (Nikon D-Eclipse C1) using EZ-C1 software.