V900502

Tissues were embedded in the optimal cutting temperature (OCT) compound and sectioned at 100 µm or 20 µm with a cryostat. Frozen sections were permeabilized and blocked in PBS containing 5% normal goat serum (SL038, Solarbio) and 0.3% Triton X-100 (V900502, Vetec) for 1 h at room temperature. The sections were then incubated with primary antibody against CD4 (1:50; ab288724, Abcam), CD8 (1:100; ab237709, Abcam), B220 (1:100; 14-0452-86, eBioscience), Laminine (1:100; MA106100, Invitrogen), P-Glycoprotein (1:72; MA1-26528, Invitrogen), GFAP (1:400; 3670S, CST), Il-1β (1:200; P420B, Invitrogen), or Fibrinogen (1:100; NBP2-80414, Novus) overnight at 4 °C. After 3 washes in PBS, sections were incubated with Alexa Fluor^® 488-conjugated anti-rabbit IgG (H + L) (1:500; 4412S, CST) and Alexa Fluor^® 555-conjugated anti-rat IgG (H + L) (1:500; 4417S, CST), or with Alexa Fluor^® 594-conjugated anti-mouse IgG (H + L) (1:500; 8890S, CST), for 1 h at room temperature. In addition, DAPI (1:2000; Sigma, 32670) was used to stain cell nuclei. Slides were mounted with Fluoro-Gel (17985-10, Electron Microscopy Sciences, Hatfield, PA, USA) and imaged using an Olympus IX71 fluorescence microscope (Olympus).

For immunofluorescence, cells plated on chamber slides were fixed with 4% Paraformaldehyde at room temperature for 15 min. Following washing three times with PBS, cells were blocked by PBS containing 0.2% Triton X-100 (Vetec, V900502) and 10% goat serum for 1 h. Then cells were incubated in primary antibody solution overnight at 4°C. The primary antibody used was rabbit anti-MAP2 antibody (1:2000, Abcam, ab281588). After three times washing with PBS, cells were incubated with donkey anti-rabbit secondary antibody (1:1000, Abcam, ab150073) for 1 h at room temperature. After washing the secondary antibody, DAPI (Alphabio, A1013) was added in the chamber for 10 min to stain the cell nuclei. The cells were examined with laser scanning confocal microscope (Zeiss, LSM880). Neurite outgrowth was evaluated for a total of 60-120 neurons per group using the Image J software, by taking 5 images containing 4-8 MAP2-positive neurons per image from triplicate samples in each of three independent experiments.

The presence of human anti-NMDAR antibodies in the serum of mice was determined using immunofluorescence staining of GluN1-transfected cells. An amount of 1 × 10⁵ HEK-293 T cells in 24-well plates on 12 mm cover-slips were transfected with 500 ng M68CT–GluN1 plasmid using 1 μL Lipofectamine™ 3000 reagent (L3000015, Invitrogen, Grand Island, NY, USA). After 48 h, cells were fixed with 4% paraformaldehyde for 30 min at room temperature, blocked with 5% BSA–0.3% Triton ™ X-100 (V900502, Vetec, Green Lane, PA, USA) for 1 h at room temperature, and then incubated with mouse serum (1:1) at 4 °C overnight and with Alexa Flour 488-conjugated goat anti-human IgG (1:500; A-11013, Invitrogen) at room temperature for 1 h. Afterward, the cells were incubated again with commercial rabbit anti-GluN1 antibody (1:200; AGC-001, Alomone, Jerusalem, Israel) at 4 °C overnight and then with Alexa Flour 555-conjugated goat anti-rabbit IgG (1:500; 4417, CST, Novi, MI, USA) at room temperature for 1 h. Cell nuclei were counterstained with DAPI (1:2000; Sigma, Cambridge, MA, USA) for 10 min at room temperature, followed by sealing and photographing using an inverted fluorescence microscope Olympus IX-71 (Olympus).