Figure 3

ExM allows three-dimensional and multicolor examination of the MPS. (a) Representative widefield images of βII-spectrin immunofluorescence before (left panel) and after expansion (middle and right panels). The middle image is a single widefield frame out of a stack made of consecutive steps in the z-axis. The right image is the corresponding frame after deconvolution processing, to eliminate out-of-focus light. Scale bars: pre-expansion 4 µm and post-expansion 1 µm. (b) Selected images from a z-stack, after deconvolution. The last panel shows a maximum projection of the shown stack. Scale bar: 1 µm. (c) x/y maximum projections of selected z-stacks are shown and color-coded for depth. The bottom panels show x/z sections #1 and #2. Asterisks indicate the depths at which crossing axons with an MPS are visible. Scale bar: 1 µm. (d) Transversal intensity profiles of double-labeled axons show the expected peripheral staining of βII-spectrin compared to the more inner staining of microtubules stained with an antibody against tyrosinated tubulin (profiles #1, and #2). Scale bar: 0.5 µm.