ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue

Abstract

We describe a clearing method for enhanced visualization of cell morphology and connections in neuronal and non-neuronal tissue. Using Clear(T) or Clear(T2), which are composed of formamide or formamide/polyethylene glycol, respectively, embryos, whole mounts and thick brain sections can be rapidly cleared with minimal volume changes. Unlike other available clearing techniques, these methods do not use detergents or solvents, and thus preserve lipophilic dyes, fluorescent tracers and immunohistochemical labeling, as well as fluorescent-protein labeling.

Fig. 1.

Rapid tissue clearing with Clear^T. (A) Fixed whole embryos (E14.5) and dissected postnatal brains (P0) were cleared overnight. The grid is visible through tissue cleared by Clear^T. (B) E14.5 embryos cleared with Clear^T or ScaleA2 reach full transparency in 1 day or 14 days, respectively. (C) Clear^T does not lead to volume changes. P0 sections (800 μm), surface area measured: pre-cleared, red line; Clear^T, blue line. (D) Clearing is reversible with PBS (30 minutes). Scale bars: 1 mm.

Fig. 2.

Retinal axon projections in brain tissue cleared with Clear^T. (A) E15.5 eye was labeled with DiI, the jaw and tongue were cut away and the head was cleared with Clear^T. DiI-labeled contralateral (C) and ipsilateral (I) retinal axons and optic chiasm are detected in both dorsal and ventral views after clearing with Clear^T. (B) Merged stack (41 images, 5 μm steps) of E14.5 DiI-labeled growth cones (GCs) (arrows) and axons (arrowheads) of the ipsilateral optic tract; imaged from the ventral surface of 200 μm brain section, before and after clearing. (C) DiI-labeled contralateral RGC projection to the thalamus and superior colliculus at E18.5. Brains were cut sagittally at the midline and cleared with Clear^T. Merged stack (51 images, 20 μm steps), viewed from the midline. DiI-labeled RGC axons in the dLGN in the thalamus (TH) and superior colliculus (SC) were undetectable in pre-cleared tissue, but easily visible after clearing. (D) CTB conjugated to Alexa Fluor 488 or 594 was injected into each eye and a 700 μm frontal section of P5 brain was cleared with Clear^T. Optical slices at 250 μm, 450 μm and 600 μm below the tissue section surface are shown (from 71 images, 10 μm steps). Both CTB labels were observable, though deeper, in cleared dLGN compared with the same tissue before clearing. Scale bars: 1 mm in C (top); 100 μm in A and bottom of C,D (bottom); 10 μm in B.

Fig. 3.

Clear^T2 clears tissue with fluorescent proteins or immunohistochemistry. (A) Clear^T cleared E14.5 actin-GFP embryos, but reduced GFP fluorescence. Formamide (50%) maintained fluorescence, but failed to clear embryos. Clear^T2 cleared embryos and maintained fluorescence. (B) P0 sections (800 μm) were transparent after Clear^T2, with no volume change. (C) P11 Thy1-GFP (M-line) hippocampus section (800 μm), before and after clearing with Clear^T2; 38 images, 20 μm steps (top and middle). GFP⁺ pyramidal neurons (arrows) and dendrites (arrowheads) in CA1 region are markedly more visible after clearing; 52 images, 2.5 μm steps (bottom). GCL, granule cell layer; ML, molecular layer. (D) Sections of E14.5 optic chiasm (200 μm), immunolabeled with the radial glial marker RC2, cleared with Clear^T2; 51 images, 3 μm steps; three optical slices shown. RC2⁺ staining was observed deeper in cleared compared with pre-cleared tissue. Blue indicates Hoechst staining. (E) E11.5 whole embryos, immunolabeled with neurofilament antibody (NF) and treated with Clear^T2. NF⁺ axons were much more visible in cleared embryos (top); magnification of trigeminal axons reaching epithelial targets (bottom). (F) Section (300 μm) of postnatal mouse brain, dLGN anterogradely labeled with CTB conjugated to Alexa Fluor 594. A single neuron was filled with biocytin and immunostained with streptavidin-Alexa Fluor 647. Clearing with Clear^T2 enhanced resolution and visibility of the dendritic arbor of the neuron. Merged stack, 55 images, 2 μm steps. CTB label is in red; biocytin-filled neuron is pseudo-colored green. Scale bars: 1 mm in A,B,E; 40 μm in C; 20 μm in D,F.