Figure 6.

Co-localization between SNAP-25 and Syntaxin 1A, detected by antibodies or nanobodies in STED microscopy. (a) Co-staining for SNAP-25 and Syntaxin 1A in primary hippocampal neurons. A polyclonal rabbit antibody against SNAP-25 and a mouse monoclonal against Syntaxin 1A were used. Rabbit anti SNAP-25 was further detected with a secondary antibody conjugated to Abberior-Star580; Syntaxin 1A primary antibody was detected with an Atto647N-conjugated secondary antibody. Nanobody co-staining was performed with S25-Nb10 conjugated to Abberior-Star580 and with Stx1A-Nb6 conjugated to Atto647N. The scale bars represent 2 µm and 500 nm in the low and high zoom, respectively. (b) Pearson´s correlation coefficients (expressed as coefficients of determination, R²) between the green and red signals within synapses were calculated from 17 independent experiments for the antibodies and from 9 independent experiments for the nanobodies (typically, 10 images per experiment were analyzed). The values are shown as box plots, with the median, 25th and 95th percentile shown in the graph, and with symbols showing outliers. The difference is significant (Wilcoxon rank sum test; p = 0.0311).