Rabbit anti synaptophysin

Cells were fixed with 4% paraformaldehyde (PFA) in PBS, permeabilized and blocked with 0.1% Triton-X100 plus and 5% normal goat serum in PBS. Then, they were incubated overnight at 4°C with the following primary antibodies: mouse anti-DLX2, 1:500 (Santa Cruz), rabbit anti-GABA, 1:1000 (Sigma), rabbit anti-GAD65/67, 1:250 (Millipore), mouse anti-GFAP, 1:200 (Millipore), mouse anti-MAP2, 1:200 (Chemicon), rabbit anti-MAP2, 1:1000 (Millipore), mouse anti-nestin, 1:200 (Chemicon), mouse anti-NeuN, 1:500 (Millipore), rabbit anti-neurofilament M, 1:200 (Millipore), mouse anti-NKX2.1, 1:1000 (Millipore), rabbit anti-OLIG2, 1:1000 (a gift from Dr. Charles Stiles, Harvard Medical School), mouse anti-PAX6, 1:250 (Millipore), mouse anti-PSD95, 1:500 (Millipore), rabbit anti-S100, 1:250 (Dako), mouse anti-SOX2, 1:500 (Millipore), rabbit anti-synaptophysin, 1:250 (Sigma), rabbit anti-TuJ1, 1:1000 (BioLegend) and mouse anti-vGAT, 1:200 (Synaptic Systems). After washing with PBS, cells were incubated with the secondary antibodies, Alexa Fluor® 555 anti-mouse IgG (Molecular Probes) and Alexa Fluor® 488 anti-mouse IgG (Molecular Probes). Cells were then counter-stained with 4,6-diamidino-2-phenylindole (DAPI) (Santa Cruz). The images were captured using a confocal laser-scanning microscope (LSM700; Zeiss) and digital inverted fluorescence microscope (DM5000B; Leica).

Imaging on fixed and permeabilized synaptosomes was carried out as previously described [88 (link),92 (link)]. The following antibodies were used: rabbit anti-synaptophysin (1:500; Sigma-Aldrich, St. Louis, MO, USA), mouse anti-GFAP (1:1000; Sigma-Aldrich), mouse anti-oligodendrocyte (RIP; 1:10,000; Millipore Corporation) and mouse anti-integrin-αM (1:25; Millipore Corporation). Anti-rabbit and anti-mouse secondary antibodies (conjugated with Alexa Fluor 488 or 633) were used (1:1000; Life Technologies Corporation, Carlsbad, CA, USA). Images were collected by means of confocal microscopy using an inverted Leica STELLARIS 8 Falcon τ-STED (Leica Microsystems, Mannheim, Germany) inverted confocal/STED microscope. A white light laser was used and optimally tuned to provide excitation of the chosen fluorochromes. Hybrid HyD detectors were used for the detection. An HC PL APO CS oil immersion objective 100× (1.40 NA) was used to collect the images, while the pinhole was set to 1 Airy size. The line scanning speed range was 400 Hz. The Leica “LAS X application Suite” software package 4.4.0.24861 was used for acquisition, storage and visualization. The purity of synaptosomal fraction was assessed by analyzing 5–7 fields from at least three different preparations.