Maximum z intensity-projected confocal image of layers 2 and 3 of the mouse somatosensory cortex after performing 20ExM and postexpansion immunostaining with antibodies to RIM1/2 (red) and PSD95 (cyan). Left, low-magnification image. Right, zoomed-in images of the three white dotted boxes ( i iii ) with separate channels for each antibody along with the merged image. The image shown is from a representative experiment using four brain slices from two mice. Brightness and contrast settings were first set by Fijis autoscaling function and then manually adjusted to improve contrast and highlight the boundary of the synapses; quantitative analysis in c f was conducted on raw image data. Scale bars are provided in biological units: 1 m (left) and 100 nm (right, iiii); AU, arbitrary units.
