Figure 4.

Proximity Ligation Assay for the Detection and Quantification of Synapses

(A–C) (A) Diagram illustrating the principle of transsynaptic PLA (left) and representative images of transsynaptic PLA signal with the described combination of antibodies used in 14 DIV cortical neurons (right; scale bar, 50 μm). (B) Composite image of 28 DIV PSD95-mVenus (ENABLED) cultures with transsynaptic PLA for Neurexin1b-Neuroligin1c and counterstained with MAP2 and Synapsin (triple stained), acquired with standard confocal microscopy (scale bar, 50 μm). Insets show different types of combinations between transsynaptic PLA spots, mVenus-PSD95, and Synapsin (center). The adjoined spot density quantification reveals differences between individual markers, pairwise combinations, and the PLA partners used (mean + SD; n = 4 wells with 15 fields/well, ∗p < 0.05, two-way ANOVA post-hoc Sidak's multiple comparisons test). (C) Maximum intensity projection from a single confocal plane of triple stained cortical culture imaged 200 times (top half), overlaid with its super-resolution result after application of super-resolution radial fluctuations (SRRF; bottom half; scale bar, 10 μm), along with magnified insets. Nearest neighbor distances were calculated between all spots of two markers, and the mean ± SD is displayed per pairwise combination; between 4 and 7 images were quantified per combination.

See Also Figures S13–S15.