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A fast, aqueous, reversible three-day tissue clearing method for adult and embryonic mouse brain and whole body

Graphical abstract

Highlights
d Fast 3D Clear is a method for large tissue clearing and 3D imaging

d The method is simple, inexpensive, and reversible

d Tissue size and endogenous fluorescence are preserved

d Fast 3D Clear enables the study of neuronal structure and connectivity

Authors
Stylianos Kosmidis, Adrian Negrean, Alex Dranovsky, Attila Losonczy, Eric R. Kandel

Correspondence [email protected] (E.R.K.), [email protected] (S.K.)

In brief
Tissue clearing enables the study of cells as units and as components of a network within intact organs. Kosmidis et al. develop an easy and speedy method for clearing large tissues and visualizing individual cells and their connections within the brain in 3D.

Kosmidis et al., 2021, Cell Reports Methods 1, 100090
November 22, 2021 ª 2021 The Authors. https://doi.org/10.1016/j.crmeth.2021.100090

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A fast, aqueous, reversible three-day tissue clearing method for adult
and embryonic mouse brain and whole body
Stylianos Kosmidis,1,3,4,* Adrian Negrean,1,3 Alex Dranovsky,5 Attila Losonczy,1,2,3 and Eric R. Kandel1,2,3,4,6,*
1Zuckerman Mind Brain Behavior Institute, Columbia University, New York, NY 10027, USA 2Kavli Institute for Brain Science, Columbia University, New York, NY 10027, USA 3Department of Neuroscience, Columbia University, New York, NY 10027, USA
4Howard Hughes Medical Institute, Columbia University, New York, NY 10027, USA 5New York State Psychiatric Institute, New York, NY 10032, USA; Department of Psychiatry, Columbia University, New York, NY 10032, USA 6Lead contact
*Correspondence: [email protected] (E.R.K.), [email protected] (S.K.) https://doi.org/10.1016/j.crmeth.2021.100090

SUMMARY

Optical clearing methods serve as powerful tools to study intact organs and neuronal circuits. We devel- oped an aqueous clearing protocol, Fast 3D Clear, that relies on tetrahydrofuran for tissue delipidation and iohexol for clearing, such that tissues can be imaged under immersion oil in light-sheet imaging sys- tems. Fast 3D Clear requires 3 days to achieve high transparency of adult and embryonic mouse tissues while maintaining their anatomical integrity and preserving a vast array of transgenic and viral/dye fluo- rophores. A unique advantage of Fast 3D Clear is its complete reversibility and thus compatibility with tissue sectioning and immunohistochemistry. Fast 3D Clear can be easily and quickly applied to a wide range of biomedical studies, facilitating the acquisition of high-resolution two- and three-dimen- sional images.

INTRODUCTION

Since tissue clearing was first described over a century ago (Spalteholz, 1914; Steinke and Wolff, 2001), several optical clearing techniques have been introduced that eliminate labor- intensive histological sectioning and facilitate studies on neuronal development, morphology, and connectivity.
Clearing methods can be categorized as organic-solvent based (i.e., 3DISCO [Erturk et al., 2012], iDISCO [Renier et al., 2014], uDISCO [Pan et al., 2016], FDISCO [Qi et al., 2019],
FluoClearBABB [Schwarz et al., 2015], PEGASOS [Jing et al., 2018]) or aqueous (i.e., CLARITY [Chung and Deisseroth, 2013], PACT-PARS [Yang et al., 2014], CUBIC [Tainaka
et al., 2014]). Organic-solvent-based protocols provide high- level tissue transparency in 3–4 days, with the exception of FluoClearBABB, which requires 10 days. The main disadvan- tages of these protocols include bleaching of fluorescent pro- tein labels (3DISCO), long antibody incubation times (iDISCO), complexity in their operation (uDISCO), toxicity of some organic

solvents, and tissue shrinkage that can impede high-resolution imaging (FDISCO). On the other hand, aqueous methods are simple in their application and can preserve fluorescent pro- teins. However, these protocols often require specific equip- ment (CLARITY), and the clearing process is lengthy (CUBIC, PACT-PARS).
We have built upon these powerful techniques to develop an alternative method of whole-tissue clearing, Fast 3D Clear. Fast 3D Clear results in highly transparent adult and embryonic mouse tissue within 3 days, requiring only four solutions and seven steps. The refractive index (RI)-matching aqueous clearing and imaging solution formulation does not produce toxic vapors and is compatible with standard microscopy and optics. The tissue morphology and size are not compromised, whereas endogenous fluorescent labels with emission spanning from blue to far red are preserved for several months. The clearing procedure of Fast 3D Clear is reversible, as tissues can be returned to their previous non-transparent state and are suitable for further processing with immunohistochemistry/immunofluorescence.

Cell Reports Methods 1, 100090, November 22, 2021 ª 2021 The Authors. 1
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

RESULTS

Fast 3D Clear achieves high tissue transparency in brains, whole adult mice, and embryos
Fast 3D Clear consists of seven steps and requires 3 days to achieve complete transparency (Figure 1A). Tissue dehydration/ delipidation relies on tetrahydrofuran (THF) (Erturk et al., 2012), which can rapidly infiltrate and conserve soft tissues (Haust, 1959). To avoid bleaching of genetically expressed reporters, we used THF at pH 9–9.5, which reduces fluorescence quenching (Qi et al., 2019). To further avoid deterioration of fluorescence and overall tissue integrity (i.e., shrinkage), as happens with the use of organic solutions (100% THF), we reversed THF-induced dehydration by gradually decreasing the THF concentration to wa- ter, leading to complete restoration of tissue size (Figures 1B and 1C). Prolonged washing of the brain with dH2O water after THF treatment causes a linear expansion of the tissue compared with its original size (Figures S1A and S1E). To maintain tissue expan- sion along with the fluorescence, we incorporated urea into the iohexol-based clearing solution (Figures 1D and S1C–S1E). We used this aqueous clearing solution to preserve and visualize the cleared tissue in an RI-matched non-toxic Cargille type A immer- sion oil with RI = 1.515. Fast 3D Clear resulted in intact, highly transparent adult mouse brains (Figures 1C and 1D) compared with fixed brains (Figures 1B and S1B). We next tested Fast 3D Clear in whole adult mice and mouse embryos. Fast 3D Clear was able to produce sufficiently transparent embryonic day (E)
18.5 mouse embryos and post-natal day (P) 24, 1-month, and 3-month adult whole mice (Figures 1E–1I), as well as whole soft or- gans (Figures S1F–S1I), while maintaining fluorescence without affecting the background (Figures S1J–S1N). Thus, Fast 3D Clear is a simple procedure that leads to high transparency in a wide variety of tissues, including the central nervous system.

Comparison of Fast 3D Clear with other clearing methods
To assess its efficiency and simplicity, we subjected brain hemi- spheres derived from the same animal to Fast 3D Clear and FDISCO, or Fast 3D Clear and RTF (Figures 1J, 1K, and S1O). We observed superior clearing by Fast 3D Clear compared with RTF and similar transparency compared with FDISCO. There was no noticeable difference in the visual transparency of the cleared tissue when transferred from the aqueous clearing solution to Cargille immersion oil (Figure 1J). However, there were considerable differences in the size of tissue cleared with Fast 3D Clear versus FDISCO (Figure 1J-L). Whereas FDISCO shrinks the tissue, Fast 3D Clear might lead to tissue expansion (Figure S1P) and therefore might provide higher magnification of the region of interest. We also used wintergreen oil as an imaging medium, with RI of 1.536 (Figure 1M). Although wintergreen oil led to tissue transparency similar to that of Cargille oil, it disinte- grated the plastic holders. Nevertheless, our results highlight the potential compatibility of Fast 3D Clear with other commercially available immersion oils with higher RI. We next tested whether there were differences between Fast 3D Clear and FDISCO in fluorescence preservation, light transmittance, and background fluorescence, using brains from GCaMP3-CaMK2-Cre trans- genic animals (Tsien et al., 1996; Zariwala et al., 2012). Using

confocal (Figures 1N, 1P, and S1R–S1S) and light-sheet micro- scopy (Figures 1O and 1Q), we measured the signal-to-noise ra- tio (SNR) by using 525/50-nm laser excitation. Although the light transmittance was similar (Figure 1R), Fast 3D Cleared hemi- spheres had a higher SNR (Figures 1S and S1Q) than the FDISCO counterparts. Importantly, Fast 3D Clear was able to preserve the fluorescence signal, similar to that obtained with traditional immunohistochemistry (Figures S1T–S1W), and the GCaMP3 signal was preserved for a year in Cargille immersion oil (Figures S1X–S1Z).

Fast 3D Clear preserves endogenous fluorescence in adult mouse brains, whole adult mice, and embryos
To test whether Fast 3D Clear can preserve the fluorescence of common fluorescent proteins, we applied it to four transgenic mouse lines: (1) Thy1-GFP-M, characterized by high levels of GFP expression in sparse neuronal populations (Ariel, 2017);
(2) GCaMP3-CaMK2-Cre (Tsien et al., 1996; Zariwala et al., 2012), in which GCaMP3 calcium-sensitive fluorescent protein is expressed in CaMK2+ neurons; (3) tdTomato-VGAT-Cre (Ka- neko et al., 2018), in which tdTomato is expressed in inhibitory neurons; and (4) cFos-CreERT2-tdTomato (Guenthner et al., 2013), in which tamoxifen administration results in tdTomato labeling of neurons active during a behavioral task. In all four transgenic lines, adult brains became completely transparent while maintaining their corresponding fluorescence. Specifically, Thy1-GFP+ neurons and GCaMP3+ CaMK2 neurons were visual- ized in the dorsal hippocampus and ventral dentate gyrus (DG) (Figures 2A–2D) and in other cortical regions (Figures S2A– S2C). The labeling intensity of inhibitory VGAT tdTomato+ neu- rons was lower in the dorsal hippocampus and in the DG compared with Thy1-GFP and GCaMP3-CaMK2-Cre (Figures 2E and 2F). After fear conditioning, tdTomato expression was visualized in cFos+ hippocampal cell bodies and dendrites (Fig- ures 2G and 2H). When Fast 3D Clear was applied to whole adult GCaMP3-CaMK2-Cre mice, we could visualize GCaMP3 in the mouse retina, olfactory bulb, and spinal cord (Figures S2D–S2F). Similarly, we applied Fast 3D Clear to E18.5 mouse embryos by using the Msx1-CreERT2-tdTomato transgenic mouse line. Confocal microscopy revealed tdTomato labeling in the eye (Fig- ure S2G) and spinal cord (Figures S2H and S2I) according to Msx1 expression (Duval et al., 2014; Monaghan et al., 1991). Because some tissues can exhibit high autofluorescence (Croce and Bottiroli, 2014), we visualized the retina (Figures S2J–S2L) and the spinal cord (Figures S2M–S2O) of GCaMP3-CaMK2- Cre mice simultaneously at 405-, 488-, and 568-nm wavelengths (with corresponding emission filters 450/25, 500/30, and 585/15) by using confocal microscopy. Fast 3D Clear resulted in mini- mum autofluorescence in the lens of the eye and in the spinal cord, and absence of signal at 405 and 568 nm. These data illus- trate that Fast 3D Clear is an optimal approach when maintaining
endogenous fluorophores is critical.

Fast 3D Clear preserves the fluorescence of synthetic and genetically encoded labels at multiple emission wavelengths To examine whether Fast 3D Clear can preserve non-endogenous fluorescence of various wavelengths, we delivered fluorescent

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Figure 1. Overview of Fast 3D Clear
(A) Schematic of the Fast 3D Clear process.
(B–D) Transparency of adult mouse brains in Cargille oil. (E–I) Whole cleared mouse embryo and adult mouse.
(J–M) Sagittal images of brain hemispheres subjected to Fast 3D Clear in (J) aqueous clearing solution, (L) Cargille oil, or (M) wintergreen oil or to (K) FDISCO in DBE.
(N–Q) Confocal (N and P) and light-sheet (O and Q) images from GCaMP3-CaMK2 paired hemispheres cleared with Fast 3D Clear (N and O) or FDISCO (P and Q).
(R)Comparison of light transmittance in brain hemispheres cleared with Fast 3D Clear and FDISCO, two-way ANOVA, F(1, 4) = 1.082, p = 0.3571.
(S)SNR of Fast 3D Clear and FDISCO cleared CaMK2-GCaMP3 hemispheres. Unpaired t test, **p = 0.0088. Scale bars, 6 mm, 200 mm, 100 mm.

Thy-GFP-M

GCaMP3-
CaMK2-Cre

tdTomato- VGAT-Cre

cFos CreERT2 tdTomato

FastBlue DG-retro

AAV1 GCaMP6

retroAAV2 Arch-tdTom

retroAAV2 IRF-670

AAV1 Flex-GCaMP6 / AAV9 Cre-tdTomato
GCaMP3/ AAV9 Cre-tdTomato

Figure 2. Fast 3D Clear preserves fluorescence in transgenic mouse brains and embryos (A–H) Confocal images showing a dorsal view of the hippocampus of (A and B) Thy1-GFP-M, (C and D) GCaMP3-CaMK2-Cre, and (E and F) VGAT-Cre-tdTomato mice, and (G and H) cFos-CreERT2-tdTomato mice after fear conditioning and tamoxifen administration.
(legend continued on next page)

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dyes or virally expressed fluorescent proteins to the DG of the adult mouse hippocampus. Intracranial administration of the retrograde neuronal tracer Fast Blue labeled nuclei in the DG and entorhinal cortex (Ent), a brain region known to provide direct inputs to the DG (Witter et al., 2017) (Figures 2I, 2J, and S2P). Administration of AAV1 CaMK2-GCaMP6 or retro-AAV2 Arch- tdTomato viruses into the DG showed that GFP and tdTomato fluorescent proteins can be preserved and visualized in intact adult mouse brains (Figures 2K–2N, S2Q, and S2R). Next, we created a retrograde virus expressing an infrared fluorophore (IRF670). Fast 3D Clear was able to preserve the infrared fluores- cence in adult mouse brains (Figures 2O, 2P, and S2S). We also measured the signal of each fluorophore from intact adult mouse brains, simultaneously, at standard excitation wavelengths (405, 488, 568, and 647 nm) with corresponding emission filters (450/25, 500/30, 585/15, and 650 IF) by using the same imaging settings for all brains. Each fluorophore generated signal restricted almost exclusively to its expected emission band (high SNR), while there was minimal fluorescence signal detected out of band through the other filters (low SNR) (Figures S2T–S2X).
Recently, Zingg et al. discovered that adeno-associated vi- ruses (AAVs) exhibit anterograde transsynaptic labeling prop- erties (Zingg et al., 2017), and can thus be used for tracing and visualizing neural circuits in a cell-type- and input-spe- cific manner. To expand the application of Fast 3D Clear in neurobiology and its utility for studying neural circuits, we in- jected a Cre-dependent AAV1-GCaMP6 (Ding et al., 2014) vi- rus into the dorsal hippocampus and a tdTomato-Cre-virus into the ventral Ent/perirhinal (Prh) cortex to assess whether an anterograde transfer of AAV will activate the Cre-depen- dent expression of GCaMP6 in the dorsal hippocampus. Three weeks later, in Fast 3D Clear-processed tissue, we observed simultaneous preservation of both GCaMP6 and tdTomato (Figures 2Q–2T). Similar results were obtained when we injected the same tdTomato-Cre adenovirus into the ventral Ent/Prh of GCaMP3 mice (Figures 2U–2X). We further applied Fast 3D Clear to CaMK2-Cre animals injected in the CA3 hippocampal subregion with a double inverted AAV encoding a designer receptor (Gq-mCherry) exclusively activated by designer drug (DREADD) technology (cloza- pine-N-oxide, or CNO) (Zhu and Roth, 2014)—a valuable tool to study the function of neuronal circuits, demonstrating the compatibility of Fast 3D Clear with the mCherry fluoro- phore (Figures S2Y and S2Z). Last, we found that Fast 3D Clear can preserve fluorescent cholera toxin subunit B (CTB). Twenty-four hours after injection into the mouse hippo- campus, CTB can be clearly traced into the Ent (Figure S2AA). The compatibility of Fast 3D Clear with synthetic and geneti- cally encoded fluorescent labels over a broad spectral range makes it an ideal method to study the structure and function of neuronal circuits.

Fast 3D Clear is compatible with light-sheet and confocal microscopy
Using light-sheet microscopy, we visualized cleared brains from the transgenic lines described above. We show that Thy1-GFP-M mouse brains and CaMK2-GCaMP6-injected brains can be imaged with sufficient resolution at the cellular level (Figures 3A–3C, Video S1). In addition, we achieved com- parable cellular resolution of GCaMP3, despite it being a weaker fluorophore than Thy1-GFP (Figures 3D and 3E). Furthermore, we visualized three-dimensionally the cleared brains of cFos- CreERT2-tdTomato and VGAT-tdTomato transgenic lines, as well as the spinal cord from Msx1-tdTomato mouse embryos (Figures 3F–3H). Using Imaris software, we visualized cleared brains in three dimensions without great compromises to morphology (Figures S3A–S3C).
We next injected mouse DG with different adenoviruses and processed the brains with Fast 3D Clear (Figures 3I–3K). In addition to the injected DG, fluorescence was preserved in retro- gradely labeled neurons, and allowed high-resolution 3D imag- ing of structures known to project to the DG, including the Ent, medial septum, mammillary bodies, and projection neurons from the contralateral DG. We also visualized individual IRF670 hilar cells, with light-sheet (Figure 3K, Video S2) and confocal mi- croscopy (Figures S3D and S3E). Moreover, we detected cFos+ in the DG (Figure S3F) and Arch-tdTomato (Figure S3G), as well as the fine processes of neurons in the CA3 region of the intact mouse hippocampus and retrosplenial cortex after GCaMP6 viral injection (Figures S3H and S3I). We also injected the IRF670 virus into the dysgranular zone (S1DZ) of the somatosen- sory cortex and visualized cortical areas projecting to S1DZ, such as the rostrolateral area (RL), anterior area (A), and antero- medial area (AM) (Figure 3L). In addition, we could trace in 3D the Ent-DG neuronal circuit by using light sheet (Figure 3M) or a spinning-disk confocal microscope (Figure S3J). To verify the specificity of the labeling, we also processed unlabeled brains of wild-type mice. We found that Fast 3D Clear resulted in mini- mum background fluorescence (Figures S3K–S3M). Finally, Fast 3D Clear allowed us to detect three-dimensionally fine neuronal processes (spines) in the insular and retrosplenial cortex of Thy1- GFP-M mouse brains, by using a 203 lens with high NA (0.95) (Figures 3N, 3O, S3N, and S3O, Video S1). In summary, Fast 3D Clear is fully compatible with both specialized and simple microscopy instruments, and it provides an efficient solution to 3D imaging of the intact mouse brain.

Fast 3D Clear is compatible with fluorescent antibody labeling
After Fast 3D Clearing, transparent mouse brains were returned to their initial opaque state (see STAR Methods). cFos-CreERT2- tdTomato-labeled and cleared brain sections were indistinguish- able from non-Fast 3D Clear processed brains in terms of both

(I–P) Fluorescence images of whole adult brains scanned at the same plane displaying the hippocampal formation (top) and the DG and Ent (bottom), from wild- type (WT) animals injected with (I and J) Fast Blue, (K and L) AAV1 GCaMP6-CaMK2, (M and N) rAAV2 Arch-tdTomato, or (O and P) rAAV2 IRF670. (Q–X) Shown in (Q)–(T) are the dorsal hippocampus and Ent/Prh labeled with GFP and tdTomato after injection of AAV1 GCaMP6 and tdTomato-Cre virus into WT mice. (Q) Hippocampal formation, (R) entorhinal cortex and dentate gyrus. (S) 10X magnification of dentate gyrus and (T) perirhinal/entorhinal cortex. Shown in (U-X) is the injection of tdTomato-Cre virus into GCaMP3 mice. (U) Hippocampal formation, (V) entorhinal cortex and dentate gyrus. (W) 10X magnification of dentate gyrus and (T) perirhinal/entorhinal cortex. Scale bars, 200 mm for 43 and 80 mm for 103 magnification.

Figure 3. Fast 3D Clear is compatible with light-sheet and confocal microscopy
(A and B) Light-sheet images from Thy1-GFP animals in (A) hippocampus and (B) cortex.
(C)Light-sheet imaging of CaMK2-GCaMP6 injection in the DG.
(D and E) CA1 region of (D) Thy1-GFP and (E) GCaMP3-CaMK2 animals.
(F–H) 3D visualization of cleared (F) cFos-CreERT2-tdTomato brain, (G) VGAT-Cre-tdTomato, and (H) spinal cord from Msx1-CreERT2-Tdtomato E18.5 mouse embryos.
(I)3D reconstruction of a whole adult mouse brain injected unilaterally with retro-AAV2 IRF670 and imaged with light-sheet microscopy (23 sagittal view).
(J)Different brain with retro-AAV2 IRF670 (top view).
(K)123 magnification image from the same brain as in (J).
(L)Injection of retro-AAV2 IRF670 into the S1DZ brain regions showing retro-labeled cells.
(M)Light-sheet 3D reconstruction from animals injected with tdTomato-Cre (EC/Prh) and GCaMP6 (DG) virus.
(N)3D reconstruction of cortical neuron from Thy1-GFP animal showing dendritic spines.
(O)Digital zoom from the same neuron in (N) showing spines. Scale bars, (A) 1,000 mm, (C) 700 mm, (F, G, J, L) 500 mm, (B, E, H, M) 200 mm, (D) 100 mm, (K, N) 20 mm,
(O) 10 mm.

Control

Ab staining

mCherry 488 tdTomato Doublecortin

Figure 4. Fast 3D Clear is reversible and compatible with antibody staining
Brain sections after reverse clearing.
(A) Sections from cFos-CreERT2-tdTomato animals incubated with only Alexa 488 and 647 secondary antibodies. Only the endogenous tdTomato can be de- tected.
(B–D) Sections from the same animals stained with antibodies against (B) mCherry (488), (C) tdTomato, and (D) doublecortin (DCX) (647). DCX staining is completely absent from the basolateral amygdala (BLA). (E–H) Confocal images from the (E) DG, (F) central gray of the pons (CGPn), (G) cerebellum, and (H) lateral septum (LSI) of Thy1-GFP half-brains stained with GFAP and TH antibodies.
(I–K) 3D visualization (light sheet) from Thy1-GFP half-brains stained with GFAP and TH.
(L) 3D visualization of cerebellum from Thy1-GFP half-brain stained with GFAP and TH using confocal microscopy.
(legend continued on next page)

tdTomato fluorescence and overall neuronal structure (Figures 4A and 4B). Fast 3D Clear did not hinder the detection of antigens, as demonstrated by the detection of mCherry in the DG (Figures 4A and 4B) and doublecortin in the DG, basolateral amygdala (BLA), and Ent/Prh (Figures 4B–4D and S4A–S4D).
To test whether Fast 3D Clear is compatible with immuno- staining in larger tissue volumes, we cleared brain hemispheres from Thy1-GFP-M mice. After detecting Thy1-GFP+ cells, we reversed the tissue transparency and subjected the samples to an iDISCO staining protocol by using antibodies against tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) (see STAR Methods). Antibody staining was followed by sample incu- bation in the Fast 3D Clearing solution until full transparency was reached. Fast 3D Clear allowed the preservation of endogenous Thy1-GFP fluorescence, as expected, as well as simultaneous tissue staining for GFAP and TH (Figures 4E–4L, Video S3). We also used GCaMP3-CaMK2 animals and subjected them to a shorter Fast 3D Clear protocol (see STAR Methods). The clearing process was reversed, and hippocampi were stained for double- cortin by using a short iDISCO protocol (Renier et al., 2016). Although iDISCO caused a small increase in the background fluorescence, we were able to visualize GCaMP3+ neurons and doublecortin+ neurons in the DG (Figures 4M–4O, S4E, and S4F). Collectively these data demonstrate that Fast 3D Clear can be compatible with iDISCO staining.

DISCUSSION

Three-dimensional imaging of cleared tissues has revolutionized neuroscience research, enabling the visualization of single neu- rons at high resolution and their assembly into circuits in the intact central nervous system. A wide variety of clearing proto- cols have become available, and several factors need to be taken into account for choosing the most appropriate approach. These deciding factors include time, cost, complexity, safety, model organism, preservation of fluorophores, and experimental question (Ariel, 2017). Here we describe Fast 3D Clear, a clearing method that is compatible with various fluorescent proteins, synthetic dyes, and antibody labeling (Table S1). The first benefit of Fast 3D Clear is speed, comparable to that of sDISCO (Hahn et al., 2019) and FDISCO. Intact adult mouse brains, peripheral organs, mouse embryos, and entire young adult mice become transparent in just 3 days. Fast 3D Clear is better suited to screening purposes, given that it is considerably faster compared with CLARITY, iDISCO, FluoClearBABB, CUBIC, PEGASOS, and PACT. Second, Fast 3D Clear is cost effective. It does not require specialized equipment, as CLARITY does, and the necessary reagents are only minimal, compared with those required by CLARITY, iDISCO, FluoClearBABB, and PEGASOS. In addition, Fast 3D Clear reagents are inexpensive and their commercially available quantities are adequate for multiple samples. Another positive feature of Fast 3D Clear is that it is simple. The protocol includes a limited number of simple incubation steps with THF, which does not require a special pro-

cess to remove reactive oxygen species and is not an immediate hazardous source if handled properly (Hahn et al., 2019; Qi et al., 2019). Fast 3D Clear also overcomes the environmental hazard of dibenzyl ether (DBE) and simplifies the clearing procedure by using an aqueous clearing solution and RI-matching alterna- tives that do not require complete dehydration with THF. Iohexol has been used previously in RI-matching solutions and as a radiographic contrast agent, and it is an ideal clearing candidate that satisfies our environmental concerns. Moreover, aqueous solutions of high RI can be prepared with ease, matching com- mon immersion oils or reaching close to the RI of DBE at a usable viscosity. Compared with some previously published methods (Jing et al., 2018; Qi et al., 2019), Fast 3D Clear completely over- comes tissue shrinkage. Reversal of the dehydration/delipida- tion THF process can restore tissue size, whereas extended incubation with water can cause tissue expansion, which can be maintained with the addition of urea without significant fluo- rescence loss. This moderate expansion feature of Fast 3D Clear can be beneficial particularly for the visualization of neuronal cir- cuits. Using Fast 3D Clear, we were able to maintain the fluores- cence of genetically encoded fluorescent proteins for more than a year and adequately maintain the fluorescence of weaker fluo- rophores such as GCaMP3. Fast 3D Clear can additionally be used in combination with antibodies to further visualize neuronal circuits. Last, Fast 3D Clear can be used to faithfully reconstruct dendrites from sparsely labeled neuronal structures.
Our future studies are to further validate Fast 3D Clear by registering the cell types in the Mouse Brain Atlas and, in parallel, perform immunohistochemistry on the same brains in order to bridge the two methods. This will provide an unprecedented strategy for registering multiple cell types in both two and three dimensions, with further applications to translational research. Last, it remains of interest to assess whether Fast 3D Clear, because of its reversibility, can be combined with other molecu- lar processes such as fluorescence-activated cell sorting and in situ hybridization. In conclusion, Fast 3D Clear can be an important and practical tool with multiple and broad applications in biochemical studies and clinical diagnoses of pathological dis- eases in the fields of neuroscience, cancer biology, and drug screening.

Limitations of the study Despite the strong advantages of Fast 3D Clear, further testing of some of its applications is still required. First, Fast 3D Clear has not been tested in organisms other than mice, for which other methods have demonstrated excellent results (reviewed in Tian et al., 2021). The second limitation of Fast 3D Clear is its modest ability to produce completely transparent hard tissues. Although other methods (e.g., BONE clear) (Jing et al., 2018; Wang et al., 2019) are extremely competent, Fast 3D Clear can be applied only to whole young adult mice with moderate bone clearing. Conversely, application of Fast 3D Clear to 3-month-old mice displays reduced efficiency in hard-tissue transparency (bones). It would be useful to test whether we can combine Fast 3D Clear

(M–O) Whole adult mouse hippocampus from GCaMP3-CaMK2-Cre animals cleared with Fast 3D Clear. (M) GCaMP3 fluorescence before staining, (N) fluorescence at 500/30 and 650LP nm for GCaMP3 and DCX in dorsal hippocampus, and (O) ventral hippocampus merged channels. Scale bars, (I, K) 500 mm, (J and L) 200 mm, (E, F, G, H, M, N, O) 80 mm, (A, B, D) 20 mm, (C) 5 mm.

with decalcification and discoloration solutions (as in PEGASOS and BONE clear) to produce a fully transparent adult mouse. Although Fast 3D Clear is compatible with iDISCO whole-tissue staining, we have not validated the stability of the antibody fluo- rescence under longer storage conditions in the Cargille oil or clearing media. We have also not used other immunostaining protocols and reagents, such as antioxidants (i.e., propyl gallate, sDISCO), to evaluate long-term storage conditions for antibody- treated tissues. Last, we have not yet confirmed the compati- bility of Fast 3D Clear with a cell registration software.

STAR+METHODS

Detailed methods are provided in the online version of this paper and include the following:

d KEY RESOURCE TABLE
d RESOURCE AVAILABILITY
B Lead contact
B Materials availability
B Data and code availability
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Animals
d METHOD DETAILS
B Light transmittance measurements
B Signal to noise ratio
B Fluorescence quantification
B Perfusion and tissue preparation
B Fast 3D Clear protocol B Fast 3D Clear solution B FDISCO clearing
B RTF clearing
B Imaging
B Immunohistochemistry
B Immunolabeling protocol (iDISCO)
B Tamoxifen (TAM)
B Contextual fear conditioning in cFos-CreERT2-tdTo- mato mice
B HEK293FT cell culture, transfection, and production of rAAV2 and AAV
d QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j. crmeth.2021.100090.

ACKNOWLEDGMENTS

We thank Dr. Carol A. Mason and Dr. Nefeli Slavi for providing us the Msx1 tdTomato embryos. The Thy1-GFP-M mouse line was a gift from Dr. Josef Go- gos. Funding was provided by the Howard Hughes Medical Institute. We also thank the Advanced Instrumentation Core of the Zuckerman Institute and particularly Darcy Peterka, Luke Hammond, and Humberto Ibarra Avila for comments on the manuscript and technical assistance.

AUTHOR CONTRIBUTIONS

S.K. and A.N. developed the clearing protocol; S.K. performed experiments; S.K., A.D., A.L., and E.R.K. designed the research; S.K. wrote the manuscript

with contributions from A.N., A.L., and E.R.K. S.K., A.N., A.L., and E.R.K. have filled an IR CU21159.

DECLARATION OF INTERESTS

The authors declare no competing interests. A patent has been filed by S.K., A.N., A.L., and E.R.K., and the Zuckerman Institute.

Received: December 18, 2020
Revised: April 13, 2021
Accepted: September 3, 2021
Published: October 11, 2021

REFERENCES

Ariel, P. (2017). A beginner’s guide to tissue clearing. Int. J. Biochem. Cell Biol.
84, 35–39.
Chung, K., and Deisseroth, K. (2013). CLARITY for mapping the nervous sys- tem. Nat. Methods 10, 508–513.
Croce, A.C., and Bottiroli, G. (2014). Autofluorescence spectroscopy and im- aging: a tool for biomedical research and diagnosis. Eur. J. Histochem. 58, 2461.
Ding, J., Luo, A.F., Hu, L., Wang, D., and Shao, F. (2014). Structural basis of the ultrasensitive calcium indicator GCaMP6. Sci. China Life Sci. 57, 269–274.
Duval, N., Daubas, P., Bourcier De Carbon, C., St Cloment, C., Tinevez, J.Y., Lopes, M., Ribes, V., and Robert, B. (2014). Msx1 and Msx2 act as essential activators of Atoh1 expression in the murine spinal cord. Development 141, 1726–1736.
Erturk, A., Becker, K., Jahrling, N., Mauch, C.P., Hojer, C.D., Egen, J.G., Hellal, F., Bradke, F., Sheng, M., and Dodt, H.U. (2012). Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat. Protoc. 7, 1983–1995.
Guenthner, C.J., Miyamichi, K., Yang, H.H., Heller, H.C., and Luo, L. (2013). Permanent genetic access to transiently active neurons via TRAP: targeted recombination in active populations. Neuron 78, 773–784.
Hahn, C., Becker, K., Saghafi, S., Pende, M., Avdibasic, A., Foroughipour, M., Heinz, D.E., Wotjak, C.T., and Dodt, H.U. (2019). High-resolution imaging of fluorescent whole mouse brains using stabilised organic media (sDISCO).
J. Biophotonics 12, e201800368.
Haust, M.D. (1959). Tetrahydrofuran (THF) for routine dehydration, clearing, and infiltration. Am. J. Clin. Pathol. 31, 357–361.
Jing, D., Zhang, S., Luo, W., Gao, X., Men, Y., Ma, C., Liu, X., Yi, Y., Bugde, A., Zhou, B.O., et al. (2018). Tissue clearing of both hard and soft tissue organs with the PEGASOS method. Cell Res. 28, 803–818.
Kaneko, R., Takatsuru, Y., Morita, A., Amano, I., Haijima, A., Imayoshi, I., Tam- amaki, N., Koibuchi, N., Watanabe, M., and Yanagawa, Y. (2018). Inhibitory neuron-specific Cre-dependent red fluorescent labeling using VGAT BAC- based transgenic mouse lines with identified transgene integration sites. J. Comp. Neurol. 526, 373–396.
Kosmidis, S., Polyzos, A., Harvey, L., Youssef, M., Denny, C.A., Dranovsky, A., and Kandel, E.R. (2018). RbAp48 Protein Is a Critical Component of GPR158/ OCN Signaling and Ameliorates Age-Related Memory Loss. Cell Rep 25, 959– 973 e6.
Monaghan, A.P., Davidson, D.R., Sime, C., Graham, E., Baldock, R., Bhatta- charya, S.S., and Hill, R.E. (1991). The Msh-like homeobox genes define do- mains in the developing vertebrate eye. Development 112, 1053–1061.
Pan, C., Cai, R., Quacquarelli, F.P., Ghasemigharagoz, A., Lourbopoulos, A., Matryba, P., Plesnila, N., Dichgans, M., Hellal, F., and Erturk, A. (2016). Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat. Methods 13, 859–867.
Qi, Y., Yu, T., Xu, J., Wan, P., Ma, Y., Zhu, J., Li, Y., Gong, H., Luo, Q., and Zhu,
D. (2019). FDISCO: Advanced solvent-based clearing method for imaging whole organs. Sci. Adv. 5, eaau8355.
Renier, N., Adams, E.L., Kirst, C., Wu, Z., Azevedo, R., Kohl, J., Autry, A.E., Kadiri, L., Umadevi Venkataraju, K., Zhou, Y., et al. (2016). Mapping of brain

activity by automated volume Analysis of immediate early genes. Cell 165, 1789–1802.
Renier, N., Wu, Z., Simon, D.J., Yang, J., Ariel, P., and Tessier-Lavigne, M. (2014). iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910.
Schmidt, E., Nilsson, O., Koskela, A., Tuukkanen, J., Ohlsson, C., Rozell, B., and Eriksson, M. (2012). Expression of the Hutchinson-Gilford progeria muta- tion during osteoblast development results in loss of osteocytes, irregular mineralization, and poor biomechanical properties. J. Biol. Chem. 287, 33512–33522.
Schneider, C.A., Rasband, W.S., and Elicieri, K.W. (2012). NIH Image to Im- ageJ: 25 years of image analysis. Nat Methods 9, 671–675.
Schwarz, M.K., Scherbarth, A., Sprengel, R., Engelhardt, J., Theer, P., and Gi- ese, G. (2015). Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains. PLoS One 10, e0124650.
Spalteholz, W. (1914). A Method for the Clearing of Human and Animal Spec- imens (S. Hirzel).
Steinke, H., and Wolff, W. (2001). A modified Spalteholz technique with pres- ervation of the histology. Ann. Anat. 183, 91–95.
Tainaka, K., Kubota, S.I., Suyama, T.Q., Susaki, E.A., Perrin, D., Ukai-Tade- numa, M., Ukai, H., and Ueda, H.R. (2014). Whole-body imaging with single- cell resolution by tissue decolorization. Cell 159, 911–924.
Tian, T., Yang, Z., and Li, X. (2021). Tissue clearing technique: recent progress and biomedical applications. J. Anat. 238, 489–507.

Tsien, J.Z., Chen, D.F., Gerber, D., Tom, C., Mercer, E.H., Anderson, D.J., Mayford, M., Kandel, E.R., and Tonegawa, S. (1996). Subregion- and cell type-restricted gene knockout in mouse brain. Cell 87, 1317–1326.
Wang, Q., Liu, K., Yang, L., Wang, H., and Yang, J. (2019). BoneClear: whole- tissue immunolabeling of the intact mouse bones for 3D imaging of neural anatomy and pathology. Cell Res. 29, 870–872.
Witter, M.P., Doan, T.P., Jacobsen, B., Nilssen, E.S., and Ohara, S. (2017). Architecture of the entorhinal cortex A review of entorhinal anatomy in rodents with some comparative notes. Front. Syst. Neurosci. 11, 46.
Yang, B., Treweek, J.B., Kulkarni, R.P., Deverman, B.E., Chen, C.K., Lubeck, E., Shah, S., Cai, L., and Gradinaru, V. (2014). Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158, 945–958.
Yu, T., Zhu, J., Li, Y., Ma, Y., Wang, J., Cheng, X., Jin, S., Sun, Q., Li, X., Gong,
H., et al. (2018). RTF: a rapid and versatile tissue optical clearing method. Sci. Rep. 8, 1964.
Zariwala, H.A., Borghuis, B.G., Hoogland, T.M., Madisen, L., Tian, L., De Zeeuw, C.I., Zeng, H., Looger, L.L., Svoboda, K., and Chen, T.W. (2012). A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo. J. Neurosci. 32, 3131–3141.
Zhu, H., and Roth, B.L. (2014). DREADD: a chemogenetic GPCR signaling plat- form. Int. J. Neuropsychopharmacol. 18, pyu007.
Zingg, B., Chou, X.L., Zhang, Z.G., Mesik, L., Liang, F., Tao, H.W., and Zhang,
L.I. (2017). AAV-mediated anterograde transsynaptic tagging: mapping corti- cocollicular input-defined neural pathways for defense behaviors. Neuron 93, 33–47.