Anti snap23

For imaging flow cytometry experiments: anti-CD8 APC-Cy7 (BD Biosciences, San Jose, CA or Tonbo Biosciences, San Diego, CA) and anti-CD56 PE-CF594 (BD Biosciences) were used for surface staining; all intracellular stains included anti-perforin D48 PE or BV421 (Biolegend, San Diego, CA) and anti-perforin δG9 FITC (Biolegend) and one of the following unconjugated antibodies: anti-CD71 (Cell Signaling Technology, Danvers, MA), anti-granzyme B (BD Biosciences), anti-rab3D (Abcam, Cambridge, MA), anti-rab4 (Pierce Antibodies, Waltham, MA), anti-rab5 (Cell Signaling Technology), anti-rab7 (Santa Cruz Biotechnology, Dallas, TX), anti-rab8 (Cell Signaling Technology), anti-rab11a (Cell Signaling Technology), anti-rab27a (Abcam), anti-rab35 (Abcam), anti-rab37 (Abcam), anti-syntaxin6 (Cell Signaling Technology), anti-syntaxin7 (R&D systems, Minneapolis, MN), anti-vti1b (Abcam), anti-VAMP3 (Abcam), anti-VAMP4 (Abcam), or anti-SNAP23 (Abcam). The unconjugated antibodies were detected using goat anti-rabbit AF647, chicken anti-goat AF647, or donkey anti-sheep AF647 secondary antibodies (Invitrogen, Carlsbad, CA). For confocal microscopy, perforin D48 FITC (Abcam), perforin δG9 AF647 (Biolegend), and goat anti-rabbit or donkey anti-sheep AF568 secondary antibodies (Invitrogen) were used, along with the selected rab or SNARE antibody.

Ipsilateral hippocampal tissues from sham and CCI injured mice were homogenized in RIPA buffer with protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and benzonase nuclease (Millipore Corporation, Billerica, MA) and mixed by rocking at 4 °C for at least 15 min. Tissues were centrifuged, supernatant was recovered, samples were diluted and standardized to protein concentrations. Protein samples were separated on 8–10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were then blocked with 5% milk or 5% BSA in 0.1 M phosphate buffer with 0.1% Tween-20 for 1 h at room temperature (RT) and incubated overnight at 4 °C with primary antibodies. Membranes were incubated for 1 h at RT with HRP-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA). Bands were visualized using SuperSignal substrate (ThermoScientific, Pittsburg, PA). The following primary antibodies were used: anti-GluR1, anti-NR1, anti-NR2B (EMD Millipore, Billerica, MA), anti-GFAP (BD Biosciences, San Jose, CA), anti-SNAP25, anti-SNAP23 (ABCAM, Cambridge, MA) and anti-β-tubulin (Sigma-Aldrich, St. Louis, MO) antibodies. ImageJ was used to perform density analysis. Protein measurements were standardized to β-tubulin and normalized to average WT sham signals.