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. 2022 Oct 31;11:e80895. doi: 10.7554/eLife.80895

Figure 2. Anti-gephyrin DARPins specifically label gephyrin at inhibitory postsynaptic sites.

Native gephyrin in fixed hippocampal neuron cultures (DIV15) probed using DARPin-FLAG clones, subsequently detected with anti-FLAG antibodies, and compared to staining with commercial anti-gephyrin antibody clone Ab7a. (A) Representative images of DARPin-FLAG clones 27B3, 27D3, 27F3, and 27G2 gephyrin puncta colocalized to Ab7a signal compared to the control DARPin E3_5. (B) Higher-magnification images of dendrite segments showing detected DARPin-FLAG signal colocalized with presynaptic VGAT. (C) Colocalization analysis indicating the fraction of gephyrin puncta that colocalize with VGAT along a proximal dendrite segment (>30 neurons/group pooled across three experiments). (D) Average puncta size identified by antibody Ab7a or DARPin-FLAG clones averaged by cell (pooled across neurons, >1100 synapses/group pooled across three experiment). Statistics: (C, D) one-way ANOVA, Tukey’s post-hoc test comparing all groups ****p<0.0001, ***p<0.0005, **p<0.005, *p<0.05.

Figure 2—source data 1. Data and statistical analysis to generate the violin plot in Figure 2C and D.

Figure 2.

Figure 2—figure supplement 1. Morphological characterization of DARPin-FLAG labeling in hippocampal neuron culture.

Figure 2—figure supplement 1.

Fixed embryonic E17 rat hippocampal neuron cultures (DIV15) were stained using DARPin-FLAG clones and subsequently detected with anti-FLAG antibodies and compared to staining with commercial anti-gephyrin antibody clone Ab7a or 3B11. (A) DARPin-FLAG control (E3_5) and clone 27D5 with no synaptic labeling. (B) DARPin-FLAG clones and antibody 3B11 that demonstrate high background labeling. (C) DARPin-FLAG clones with highly specific inhibitory synapse labeling.