Anti synapsin 1

Fixed brains were paraffin-embedded and cut into 4-μm sections. After antigen retrieval, the sections were blocked and stained with anti-Oligo2 (1:500, Proteintech), anti-MBP (1:200, Abcam), anti-NG2 (1:100, Millipore), anti-Iba1 (1:500, Proteintech), anti-GFAP (1:500, Proteintech), anti-MAP2 (1:500, Proteintech), anti-PSD95 (1:500, Cell Signaling Technology), or anti-Synapsin I (1:500, Cell Signaling Technology). Then, the sections were incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (1:500, Abbkine, China) or (HRP)-conjugated anti-mouse antibody (1:500, Abbkine, China). The sections were developed using DAB peroxidase substrate (Beyotime Biotechnology, China). The sections were photographed with an Olympus AH-2 light microscope (× 200; Olympus). Images were imported into ImageJ for quantification. Briefly, the well-stained area on the images was first selected; then, the mean integrated optical density value of the area was calculated by ImageJ. According to the mean integrated optical density of the control group, the relative density of the EAE group and the MCC950 treatment group was calculated.

The following antibodies were used for western blot: Anti-neuroserpin goat polyclonal antibody G64 (generation and affinity-purification have been previously described^{13 (link)}) (0.5 ug/ml); Anti-synaptophysin (Abcam ab32594, 1:1000); Anti-SNAP25 (Abcam ab41455, 1:1000); Anti-neuroserpin (Abcam ab46761, 1:5000 and ab32901, 1:2000); Anti-synapsin-I (Cell Signaling Technology D12G5, 1:1000); Anti-PSD-95 (Millipore clone EP2652Y, 1:1000); Anti-beta-actin (Millipore clone C4, 1:5000); Anti-PDI (StressMarq SPC114C, 1:2000); Anti-GFP (Clontech mouse Living Colors monoclonal antibody 632,459, 1:5000). The following antibodies were used for immunofluorescence: Anti-MAP2 (Sigma M4403, 1:200); Anti-cleaved caspase 3 (R&D Systems AF835, 1:100); Anti-synaptophysin (Abcam ab32594, 1:200); Anti-PSD-95 (Millipore MAB1598, 1:100); Anti-neuroserpin goat polyclonal antibody G64 (3 ug/ml).

The rodents were deeply anesthetized and euthanized by decapitation. The bilateral PFC regions were dissected out immediately and homogenized by sonic disruption. For cultured primary cells, after 4 hours drug treatment, the cells were harvested. The next steps for PFC tissues and primary cells were the same. The whole homogenate or cell lysate was centrifuged at 14,000 × g for 10 min, and the total protein concentration was assessed. The proteins were separated on SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked and then incubated with primary antibody (anti-mTOR, 1:1000, Cell Signaling; anti-pmTOR (Ser 2448), 1:1000, Cell Signaling; anti-pPKA Cα (Thr197), 1:1000, Cell Signaling; anti-PKA Cα, 1:1000, Cell Signaling; anti-PSD 95, 1:1000, Invitrogen; anti-GluR1, 1:1000, Abcam; anti-synapsin I, 1:1000, Cell Signaling; or anti-tubulin, 1:1000, Cell Signaling) at 4 °C overnight. The membrane was washed and incubated with secondary antibody (goat anti-Rb IgG, 1:5000, Abcam). The membrane was washed, chemiluminescence (ECL) reaction solution was added, and then the samples were exposed to X-ray film (Carestream) for visualization.

Synaptosomal lysates (20μg per lane) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes, incubated with anti-CaMKII (1:10,000, Cell Signaling, Beverly, MA, USA), anti-p-CMKII (1:2000, Cell Signaling, Beverly, MA, USA), anti-synapsin I (1:30,000, Cell Signaling, Beverly, MA, USA), and anti-p-synapsin I (Serine 603) (1:2000, GeneTex, Irvine, CA, USA) antibodies for overnight at 4 °C. This was followed by washing with Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubation with horseradish peroxidase-conjugated secondary antibodies (1:5000, Cell Signaling, Beverly, MA, USA) for 2 h at room temperature. Immunoreactive bands were visualized by chemiluminescence (GeneTex, Irvine, CA, USA). Quantification was obtained by scanning densitometry of five independent experiments, and analyzed with Image J software (version 1.43, Bethesda, MD, USA) [30 (link)].