Supplementary Figure 12: ScaleSQ(0) and ScaleSQ(5): two quick versions of ScaleS applicable to brain slices

(a–h) ScaleSQ(0).

(a–e) Fluorescence (a and e) and transmission (b–d) images of a 1-mm-thick brain slice prepared from an 8-week-old YFP-H mouse before (a) and after 0- (b), 1- (c), and 2- (d and e) hour incubation in ScaleSQ(0) at 37 °C. Similar observation was performed using 9 other slices.

(f–h) TEM observation of a brain sample restored from ScaleSQ(0). Ultrathin sections were prepared and imaged in the same way as for Supplementary Fig. 6. (f) A micrograph containing a neuronal soma. (g and h) Magnified micrographs corresponding to the boxes in (f), showing an excitatory, asymmetric synapse, and a myelinated axon, respectively. Similar ultrastructure was observed for other 25 excitatory synapses and 9 myelinated axons.

(i–p) ScaleSQ(5).

(i–m) Fluorescence (i and m) and transmission (j–l) images of a 1-mm-thick brain slice prepared from an 8-week-old YFP-H mouse before (i) and after 0- (j), 1- (k), and 2- (l and m) hour incubation in ScaleSQ(5) at 37 °C. Similar observation was performed using 4 other slices.

(n–p) TEM observation of a brain sample restored from ScaleSQ(5). Ultrathin sections were prepared and imaged in the same way as for Supplementary Fig. 6. (n) A micrograph containing a neuronal soma. (o and p) Magnified micrographs corresponding to the boxes in (n), possibly showing an excitatory, asymmetric synapse, and a myelinated axon, respectively. Similar ultrastructure was observed for other 9 excitatory synapses and 4 myelinated axons.

(g,o) Solid arrowheads indicate thickened postsynaptic densities. Den, dendrite; AT, axon terminal.

(h,p) Ax, axon; My, myelin.

(q) Change in size of slices after PBS(–)-, ScaleSQ(0)-, and ScaleSQ(5)-treatment. Four slices were used for each method. Three distances on each slice were measured, and the values after treatment were normalized to the initial values. Mean ± s.e.m. (n = 12).

Scale bars: a–e, i–m, 5 mm; f, n, 4 μm; g, h, o, p, 200 nm.