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. 2016 Mar 16;7:10773. doi: 10.1038/ncomms10773

Figure 4. Comparison of mSA and GFP nanobody.

Figure 4

(a) DIV 15 axons expressing GFP-Nrx1β were live-labelled using anti-GFP nanobody-Atto647, followed by immunolabelling for VGlut1 to stain pre-synaptic glutamatergic terminals. The merged image shows the colocalization between GFP-Nrx1β (red) and VGlut1 (green). (b) Schematics showing the labelling of AP-Nrx1β with mSA-Atto594 (top) and GFP-Nrx1β with GFP nanobody-Atto594 (bottom). (c) Examples of axonal regions from DIV 15 neurons expressing EGFP, AP-Nrx1β and BirAER (top); or GFP-Nrx1β (bottom). From left to right: GFP signal, Nrx1 β detection maps, colour-coded Nrx1β trajectory maps (green: fast-diffusing pool, that is, D>0.01 μm2 s−1; magenta: slow diffusing pool, that is, D<0.01 μm2 s−1), merged image showing Nrx1β trajectories overlaid with GFP (grey). (d) Distributions of the diffusion coefficients obtained for AP-Nrx1β or GFP-Nrx1β (mSA, n=6; GFP nanobody, n=5 cells from two different experiments). (e) Schematics showing the structure of the AMPA receptor auxiliary protein stargazin (Stg) with the insertion of an AP tag in the first extracellular protein loop. (f) Example of DIV 15 neurons co-expressing the synaptic marker Homer1c-GFP, AP-Stg and BirAER. AP-Stg individual molecules were tracked using mSA-Atto594 by uPAINT. Super-resolved AP-Stg localization and trajectory maps were reconstructed from 4,000 images of 20-ms exposure time, with the same colour code as in b. (g) Diffusion coefficient distribution of AP-Stg inside and outside synapses (n=6 cells from two different experiments).