Supplementary Figure 2: Characterization of macroscopic structures of brain samples during and after ScaleS-treatment

(a) Three and two fixed whole mouse brains (9 weeks old) were treated with ScaleS (Supplementary Fig. 1b) (red lines) and PBS(–) (black lines), respectively. Sample volume was measured by liquid displacement (Ref. 2) throughout the protocol. ScaleS0 reduced the volume of a fixed whole mouse brain to approximately 55% of the original in approximately 6 hrs. Then, the urea-containing and salt-free ScaleS (ScaleS1, ScaleS2, and ScaleS3) solutions gradually cleared the sample with moderate expansion. After treatment with ScaleS3, the sample expanded to approximately 150% of the original volume. The deScaling before ScaleS4 incubation shrank the sample to approximately 90% of the original. After equilibration in ScaleS4, the volume returned to nearly 100% of the original (102.1 ± 3.5%, mean ± s.d., n = 3).

(b) Comparison of macroscopic structures in 2-mm-thick brain slices before and after ScaleS treatment.

A horizontal slice (left) and a coronal slice (right) both containing the hippocampus were prepared from YFP-H mice (21 weeks old). Slices were imaged using a fluorescence stereomicroscope for transmission (T) and fluorescence from YFP. The transmission images before and after the treatment were traced and colored blue and red, respectively (T trace). The blue image was superimposed on the red one to create a merged image.

In both merged images (horizontal and coronal), blue and red signals coexist almost everywhere, indicating that the ScaleS method is free from a tissue deformity problem. Scale bars: 5 mm.

(c) A whole mouse brain of a YFP-H mouse (13 weeks old) was fixed and cleared by ScaleS. After incubation in ScaleS4 solution for 15 months at 4 °C, a transmission image (T) and a fluorescence image (YFP) were taken using a fluorescence stereomicroscope. Scale bars: 5 mm.