Figure 2

Comparison of clearing time, tissue size changes, and protein loss with eight protocols. (A) Optical transparency comparison of half of the 1 mm adult mouse sagittal blocks from eight different protocols cleared for different durations and after refractive index matching in FocusClear. (B) Box-whisker plots of tissue weight before and after clearing in each protocol, showing the smallest change with the FACT protocol (*p < 0.05; **p < 0.01; ***p < 0.001). (C) The means and standard errors of the weight change ratios in the brain sections for the eight groups are shown. There was an increase in weight in the hydrogel-based protocols and at higher temperatures. (D) Box-whisker plots of tissue area changes before and after clearing with each protocol and after refractive index matching with FocusClear. The smallest changes were seen for the FACT protocol and the SDS4%-37 °C protocol (*p < 0.05; **p < 0.01; ***p < 0.001). (E) Comparisons of means and standard errors of the tissue area change ratio across the eight groups. (F) Comparisons of daily and total (T) tissue protein loss measurements in the eight protocols. There was a reduction in protein loss at low temperatures. The arrowheads point to the day that transparency was achieved with each protocol.