Figure S5. Validation of AAV-mediated... | Rockefeller University Press

Figure S5.

$Validation of AAV-mediated MRLC mutants expression in mouse cortex and analysis of axon fragmentation in vitro and in vivo. (A) Strong mechanical stress (200 μl/min, 180 s) was applied to axons of AAV-infected neurons expressing GFP and MRLC mutants in the AoC. At 24 h after flux, neurons were fixed and used for fragmentation analysis. Representative images depict the morphology of stressed axons, presenting raw images of the GFP channel (left) and extracted fragmented fractions (right). Fragmentation extraction criteria are based on circularity >0.8 and area between 0.1 and 30.0 μm2. Bar = 30 μm. (B) Quantification of the ratio of fragmented axons (N = 41, 24, 32, 28). (C) Confocal images show AAV-mediated overexpression of GFP and MRLC mutants in mice cortex, detected using MRLC antibody 14 days after viral injection. Arrowheads indicate neurons overexpressing MRLC. Bar = 0.4 mm (left), 0.1 mm (right). (D) 3D stacks of confocal microscopy in brain slices from a sham mouse reveal the continuous morphology of individual commissural axons. Bar = 100 μm (left), 20 μm (right). (E) Representative images for Imaris detection of axon fragmentation; the magnified ROIs were shown in Fig. 8 H. Sparsely (1#) and densely (2#) labeled ROI locations were indicated on these uncropped images by yellow boxes. Bar = 100 μm. Data represent mean ± SEM; unpaired two-tailed Student’s t test; *P < 0.05.$

Validation of AAV-mediated MRLC mutants expression in mouse cortex and analysis of axon fragmentation in vitro and in vivo. (A) Strong mechanical stress (200 μl/min, 180 s) was applied to axons of AAV-infected neurons expressing GFP and MRLC mutants in the AoC. At 24 h after flux, neurons were fixed and used for fragmentation analysis. Representative images depict the morphology of stressed axons, presenting raw images of the GFP channel (left) and extracted fragmented fractions (right). Fragmentation extraction criteria are based on circularity >0.8 and area between 0.1 and 30.0 μm². Bar = 30 μm. (B) Quantification of the ratio of fragmented axons (N = 41, 24, 32, 28). (C) Confocal images show AAV-mediated overexpression of GFP and MRLC mutants in mice cortex, detected using MRLC antibody 14 days after viral injection. Arrowheads indicate neurons overexpressing MRLC. Bar = 0.4 mm (left), 0.1 mm (right). (D) 3D stacks of confocal microscopy in brain slices from a sham mouse reveal the continuous morphology of individual commissural axons. Bar = 100 μm (left), 20 μm (right). (E) Representative images for Imaris detection of axon fragmentation; the magnified ROIs were shown in Fig. 8 H. Sparsely (1#) and densely (2#) labeled ROI locations were indicated on these uncropped images by yellow boxes. Bar = 100 μm. Data represent mean ± SEM; unpaired two-tailed Student’s t test; *P < 0.05.