Figure 1.

Not All Synapse Markers can be Used for Synapse Quantification

(A–D)(A) Markers of different functional classes considered in this work (adapted from Servier medical art); (B) Quantification of the spot density after immunostaining yields variable results even between antibodies that target the same marker (mean + SD, n = 6 wells with 15 fields/well, ∗p < 0.05, one-way ANOVA post-hoc Sidak's multiple comparisons test). (C) The auto-correlation function (ACF) allows segmentation-independent evaluation of staining performance, by calculating the Pearson's correlation coefficient between an image and its duplicate as a function of a lateral shift (sliding window). The ACF amplitude and spread report on the SBR and the variability across images, respectively; the full width at half maximum (FWHM) correlates with spot size. A crisp synapse spot image has a high amplitude (>0.75) with narrow spread (<0.08) and small FWHM (≤11), whereas a noisy image has a small amplitude with a large standard deviation and large FWHM. (D) Scatterplot of ACF parameters for the different synapse antibodies tested on primary cortical cultures at 14 DIV (n = 90 images originating from 6 wells), along with representative images for a selected subset (scale bar, 10 μm). Inset shows which combinations are retained (in color) after gating for optimal parameter settings.

See Also Figures S1–S4.