Figure 7. Amount of rapsyn-GFP positive punctate structures and their co-localisation with BGT-AF647 are increased in FLAG-MCLT or silMyoVa transfected muscles in vivo.

TA muscles of adult wildtype mice were either transfected with rapsyn-GFP (E–F) or co-transfected with rapsyn-GFP and FLAG (A–D), FLAG-MCLT (A–D) or silMyoVa (E–F). Muscles in E–F were also injected with BGT-AF647 during transfection. Ten days later, muscles were locally injected with BGT-AF647 (A–D) or BGT-AF555 (E–F) and imaged with in vivo confocal microscopy. A–B: Overlay images, rapsyn-GFP (green), BGT-AF647 (red), co-localising signals (yellow). Scale bar, 50 µm. A: Maximum z-projections of 80 (FLAG) and 37 (FLAG-MCLT) optical slices taken at 1.5 and 1 µm interslice distance, respectively, encompassing the entire extensions of shown NMJs in depth. B: Single optical sections binarised with equal thresholds upon background subtraction for better visibility. Rapsyn-GFP vesicular structures positive and negative for BGT-AF647 in the overlay image, white-filled and empty arrowheads, respectively. C: Quantification of rapsyn-GFP vesicular structures per microscopic field (≥12 per condition). Data, mean±s.e.m. (n = 3 mice). D: Quantification of BGT-AF647 positive and negative rapsyn-GFP vesicular structures. Data, mean±s.e.m. (n = 3 mice, ≥12 microscopic fields per condition, 1112 (FLAG) and 3982 (FLAG-MCLT) analysed vesicular structures). E–F: Overlay images, rapsyn-GFP (green), BGT-AF555 (red), BGT-AF647 (blue). Scale bars, 25 µm. E: Maximum z-projections of 33 (control) and 53 (silMyoVa) optical slices taken at 0.8 and 1 µm interslice distance, respectively, encompassing the entire extensions of shown NMJs in depth. F: Single optical slices binarised with equal thresholds upon background subtraction for better visibility through the centre of shown NMJs. Overlay of rapsyn-GFP and BGT-AF555 (yellow), of BGT-AF555 and BGT-AF647 (magenta), and of rapsyn-GFP, BGT-AF555 and BGT-AF647 (white).