Mouse anti synaptophysin

For the neuron staining, we followed the methods of Zhang et al. [18 (link)]. For colocalization, cortical neurons on coverslips were fixed with 4% paraformaldehyde for 10 min after a brief rinse in prewarmed ECS at room temperature, then permeabilized, and blocked through incubating in PBS containing 0.5% Triton X-100 and 5% BSA for 0.5 hr. Then, the neurons were incubated with primary antibodies to SIL1 (rabbit anti-SIL1, Abcam) and synaptophysin (mouse anti-synaptophysin, Abcam) or SD95 (mouse anti-PSD95, Abcam) in PBS containing 0.5% Triton X-100 overnight at 4°C. After rinsing in PBS containing 0.5% Triton X-100 3 times, neurons were incubated with both Alexa 488-conjugated secondary antibody and Alexa 546-conjugated secondary antibody (donkey anti-rabbit or mouse secondary antibody, Abcam) for 1 h at room temperature. After rinsing with PBS three times, neurons were examined under a 60x, 1.4 numerical aperture oil-immersion objective on an Olympus confocal microscope.

2-, 4- and 6-month EFAD mice were anesthetized with sodium pentobarbital (50 mg/kg), transcardially perfused with ice-cold PBS, brains removed and dissected into cortex and hippocampus, snap-frozen in liquid nitrogen and stored at -80°C, as previous described [133 (link)]. Dissected brains were homogenized in lysis buffer [90 (link),132 (link)] (50 mM Tris-HCl, 150 mM NaCl, pH7.4, 1% Triton X-100, 1x protease inhibitor cocktail) and 40 μg of total protein (BCA protein assay kit; Pierce, Rockford, IL) was separated on 4–12% gradient Bis-Tris gels (Invitrogen) under reducing conditions, and transferred to PVDF membranes [47 (link)]. The following primary antibodies were used: rabbit anti-PSD95 (1:3000, Abcam), mouse anti-synaptophysin (1:2000, Abcam), mouse/rabbit anti-β-actin (1:2000; Abcam), rabbit anti-drebrin antibody (1:1000; Abcam), rabbit anti-NMDAR1/anti-NMDAR2B (1:1000; Millipore), anti-NMDAR2A (1:500; Millipore), mouse anti-apoE (1:600; Santa Cruz), rabbit anti-BDNF (1:200; Santa Cruz), rabbit anti-p-CaMK-II (1:1000; Santa Cruz) and rabbit anti-p-CREB (1:1000; Cell Signaling) [90 (link),132 (link)]. HRP-conjugated secondary antibodies, enhanced chemiluminescence (Amersham, Piscataway, NJ) and Image J software were used to quantify densities of the immunoreactive bands relative to β-actin.

Rats were deeply anesthetized and perfused intracardially with PBS followed by 4% paraformaldehyde/PBS (pH 7.4). The dorsal horn (L4–5) samples were removed, post-fixed in the same fixative (4 °C for 4 h) and cryoprotected in 30% sucrose solution for overnight at 4 °C. Transverse lumbar spinal cord sections (30 µm) were cut using a cryostat and mounted on glass slides. The sections were pre-incubated with 5% BSA 1 h in PBS to block non-specific binding. Subsequently, the sections were incubated with rabbit anti-Munc13-1 (rabbit, 1:200, Synaptic Systems, Goettingen, Germany), together with mouse anti-synaptophysin (a presynaptic marker, 1:500, Abcam, Cambridge, USA), or mouse anti-homer1 (1:500, Abcam, Cambridge, USA) overnight (4 °C). After three times of rinsing with PBS, spinal sections were incubated at 37 °C for 1 h with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:1500; Invitrogen, Grand Island, NY) as well as with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1500; Invitrogen, Grand Island, NY). The spinal sections were subsequently rinsed in PBS, and coverslips were applied. When these fluorescent markers were excited, they were easily detected by a camera-coupled device (X-plorer; Diagnostic Instruments, Inc., USA) through a fluorescent microscope (LEICA DM2500, Germany).