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. Author manuscript; available in PMC: 2017 Jan 29.
Published in final edited form as: Cell. 2015 Dec 3;163(6):1500–1514. doi: 10.1016/j.cell.2015.11.025

Figure 1. Synchronizing Dialdehyde-tissue-gel Formation Enables Scalable Tissue Preservation.

Figure 1

(A) Chemical structures of various multifunctional fixatives. (B) Crosslinked protein gels before and after exposure to the elution condition. Scale bars, 10 mm. Polyacrylamide (AA) gel swelled and became fragile, whereas multifunctional fixative gels remained intact with minimal expansion. (C) Mass percent change of crosslinked protein gels after exposure to the harsh condition. EDGDE, GE21, and EX-313 were incapable of forming gels at low BSA concentration. Error bars show mean ± SD. (D) The gelation time for protein gels crosslinked with GA is nearly 200-fold higher at pH 3 than it is at neutral pH at 4°C. Error bars show mean ± SD. (E) Schematic diagram illustrating the process of scalable and uniform tissue-gel formation without perfusion using SWITCH. GA molecules diffuse into an intact tissue without reacting with biomolecules in pH 3 buffer (SWITCH-Off step). When GA is uniformly dispersed throughout the tissue, the sample is moved to pH 7 buffer (SWITCH-On step) to initiate global gelation/fixation and achieve uniform tissue preservation. (F) Coronal slices from the middle of whole rat brains passively fixed with (bottom) or without (top) SWITCH. After fixation, the middle coronal slices were cut and incubated in the elution condition for 1 hr. The core of the control slice completely disintegrated, whereas the SWITCH-processed slices remained intact. Scale bars, 6 mm. (G) Only ~3% of proteins are lost in SWITCH-processed brain tissues as opposed to ~10–30% with AA-based methods. Error bars show mean ± SD. (H and I) Antigenicity of proteins is well preserved throughout the clearing process in SWITCH. Of the antibodies tested, 86 of 90 are compatible with SWITCH. (J and K) SWITCH-mediated fixation maximally preserves macroscopic (J) and microscopic (K) structures throughout the elution process. (J) Cross-sectional images of 1-mm-thick mouse coronal slices after exposure to the elution condition. The CLARITY-processed tissue shows significant tissue deformation and collapse, whereas the SWITCH-processed tissue is highly uniform with no signs of macroscopic deformation. Z-step size, 20 μm; 10×, 0.3 NA, water-immersion objective. Scale bars, 1 mm. (K) GFP-expressing neurons in the cortex of Thy-1-EGFP mouse brain before and after exposure to the elution condition and anti-GFP staining. 25×, 0.95 NA, water-immersion objective. Scale bars, 30 μm. See also Figures S1 and S4, and Table S1.