Quantifying Synaptic Proteins by ELISA

SYP and NSE were measured by sandwich ELISA and PSD95 by indirect ELISA [52 (link), 62 (link)]. The capture antibody, SYP (Abcam, Cambridge, UK) or NSE (Enzo Life Sciences, Exeter, UK), was diluted 1:1000 in coating buffer and the wells preincubated overnight at 4 °C. Blocking buffer (1% BSA-PBS) was added for 1 h followed by the load in duplicate of either serial 5-fold dilutions of recombinant NSE protein (0.008–5 μg/ml; Abcam) to generate a standard curve, or 2-fold dilutions of recombinant SYP protein (0.34–5.5 μg/ml; Abnova, Taipei City, Taiwan), homogenates at a 1:10 or blanks. Two hours later, peroxidase-labelled, mouse monoclonal anti-NSE (Abcam) or biotinylated anti-mouse IgG for SYP detection (Vector Laboratories), was added and incubated in the dark for 2 h.