Fig. 2

Increased sensitivity of antibodies applied on CLARITY-processed sections. a Cleared (orange) and non-cleared (blue) frontal cortex sections from case p1 were stained for CAMKIIA and imaged 15 µm deep in 1.8 µm steps using a confocal microscope. Z-level I (z = 0 µm), III (z = 3.6 µm), and VI (z = 9 µm) are provided as single images (left). Mean intensities of each z-step were exported with LAS-X software from two stacks (cortex layer V), and the mean was plotted in dependency of the z-level (right). A side view (xz scan) is shown in the middle. Scale bars, 10 µm. b CAMKIIA-positive surfaces in the neuropil outside dendrites of cases p1 (perfusion; filled circle) and p2 (white circle) were counted in z-levels I (z = 0 µm), III (z = 3.6 µm), and VI (z = 9 µm) of two z-stacks per case and condition (acquired with identical settings). In total, two regions of interest (ROIs) were analyzed per image in Imaris with constant parameters for plus and minus CLARITY per z-plane. The mean ratios between plus and minus CLARITY (y axis) of two technical replicates are plotted; a ratio of 1 (black line) indicates identical surface numbers for plus and minus CLARITY. c Mean intensity of GLUA2 staining on case p1 was plotted in dependency of the z-level. Acquisition strategy is the same as outlined in panel (a). Scale bars, 5 µm. d More sensitive detection of GLUA2 puncta after CLARITY, analysis as described in panel (b)