S5768

Antibodies used included anti-GFP (A11122, 1:1000, Invitrogen) to label transfected cells and neuronal morphologic features. The antibodies used for Western blot analyses include: anti-BDNF (ab108319, 1:3000, Abcam), anti-FLAG M2-peroxidase conjugated (1:20,000, Sigma), anti-GFP-peroxidase conjugated (clone 3D8A1B8, 1:10,000, Abking), anti-MBNL1 polyclonal antibody (ABE241, 1:1000, Millipore) [21 (link)], anti-DIC (dynein intermediate chain)(ab23905, 1:1000, Abcam), anti-DYNLL2 (Merck, ABT140), anti-PSD95 (post-synaptic density 95, clone 3H4.3, 1:2000, Millipore), anti-synaptophysin (S5768, 1:1000, Sigma), anti-TrkB (#4603, 1:1000, Cell Signaling), anti-phospho-TrkB (Y817) (MA5-32,207, 1:1000, Invitrogen), anti-HSP90 (heat shock protein 90, a gift from Dr. Yi-Ping Hsueh, 1:2000) and anti-GAPDH (MAB374, 1:20,000, Millipore). Anti-GFP antibodies (clones 19C8 and 19F7) used in immunoprecipitation were from Memorial Sloan-Kettering Monoclonal Antibody Facility. To detect the level of BDNF, the nitrocellulose membrane underwent antigen retrieval to boost the signal [22 (link)].

mouse anti-β-III-tubulin (Sigma #T8660; 1:2000), mouse anti-microtubule-associated protein 2a+b (MAP2, Sigma #M1406; 1:2000), rabbit anti-tyrosine hydroxylase (TH, Millipore #AB152; 1:600), mouse anti-synaptophysin (Sigma #S5768; 1:200), goat anti-forkhead box A2 (FOXA2 (Sigma #AF2400; 1:250), rabbit anti-GABA (Sigma #A2052; 1:2000), rabbit anti-GFAP (DAKO #Z0334; 1:4000), rabbit anti-TOM20 (Santa Cruz #SC-11415, 1:1000), goat anti-HSP60 (Santa Cruz Biotechnology #SC-1052, 1:200).

After extraction, purified synaptosomes were processed as in Sokolow [41 (link)]. Primary antibodies used were anti-Synaptophysin 1 : 500 (#S5768, Sigma) and anti-p-JNK 1 : 100 (#6254, Santa Cruz) while fluoresceinated secondary antibodies were Alexa Fluor 488 anti-Mouse and Alexa Fluor 594 anti-Rabbit antibodies. Images were acquired with Super-Resolution microscope (N-SIM, Nikon-Structured Illumination Microscopy). Protein colocalization was analysed using Pearson's colocalization coefficient with Imaris software.

For single iEM analysis, frozen sections from MCAO and intact mice were incubated with a primary rabbit anti-GFAP antibody (1:200, Dako, Z0334) for 3 days at 4 °C followed by incubation with a nanogold-conjugated anti-rabbit secondary antibody (1:100; Invitrogen, N-24916) for 1 day at 4 °C. For double iEM, the sections were incubated with mouse anti-synaptophysin (1:500; Sigma, S5768) and rabbit anti-GFAP antibodies for 4 days at 4 °C followed by incubation with nanogold-conjugated anti-mouse (1:100; Invitrogen, A-24921) and HRP-conjugated anti-rabbit (1:500; Jackson ImmunoResearch, 111-035-144) antibodies for 1 day at 4 °C, and visualized with DAB solution (3,3′-diaminobenzidine tetrahydrochloride; Wako, 040-27001) with H₂O₂. After fixation with 2.5% glutaraldehyde for 10 min and silver enhancement with the HQ-Silver Kit (Nanoprobes Inc.) for 10 min at 25 °C in the dark, the sections were post-fixed with 1% OsO₄, dehydrated through an ethanol gradient (50–100%), followed by acetone and QY1 (butyl glycidyl ether), and embedded in Epon. Ultrathin sections (70 nm thick) were prepared and stained with 2% uranyl acetate and 80 mM lead citrate for 15 and 10 min, respectively. The sections were observed under a transmission EM (JEOL model 1230), and images were collected using the Digital Micrograph 3.3 (Gatan Inc.).

To investigate the immunoreactivity of Aβ accumulation, the retina and brain tissues were incubated using a blocking solution (0.05% bovine serum albumin, 1.5% normal goat serum, and 0.3% Triton X-100 in PBS) and mouse anti-4G8 antibody specific for Aβ_17-24 (1:2000; BioLegend, San Diego, CA, USA) overnight at 4 °C. To examine the immunoreactivity of neuronal nuclei (NeuN) and synaptophysin (SYN), free-floating brain sections were incubated overnight at 4 °C with the mouse anti-NeuN antibody (1:1000; Cat.# MAB377, Merk KGaA, Darmstadt, Germany) or mouse anti-SYN antibody (1:500; Cat.#S5768, Sigma-Aldrich), respectively, in blocking solution. Additionally, the retina and brain tissues were incubated with donkey Alexa 488-conjugated anti-mouse IgG (1:200; Thermo Fisher Scientific Inc., Waltham, MA, USA) or donkey Alexa 594-conjugated anti-mouse IgG (1:200; Thermo Fisher Scientific Inc.) for 1 h at room temperature. To counterstain the nuclei, the retina and brain tissues were mounted on ProbeOnTM Plus Microscope Slides (Thermo Fisher Scientific Inc.) and covered with Fluoroshield^TM with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich).