EZ Clear for simple, rapid, and robust mouse whole organ clearing
- Department of Integrative Physiology, Baylor College of Medicine, United States
- Optical Imaging and Vital Microscopy Core, Advance Technology Cores, Baylor College of Medicine, United States
- Department of Education, Innovation and Technology, Baylor College of Medicine, United States
- Cardiovascular Research Institute, Baylor College of Medicine, United States
- Department of Neurosurgery, Baylor College of Medicine, United States
Figures
Figure 1 with 1 supplement
EZ Clear is a simple, rapid, and efficient tissue clearing process.
(A) The three steps, requiring 48 hr of the EZ Clear tissue clearing protocol. (B) Brightfield representative images of adult mouse brains (9 weeks of age) fixed in 4% paraformaldehyde (PFA) only, …
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Figure 1—source data 1
Quantitative comparison of the size changes of the mouse brains before and after processing with 3DISCO, Fast 3D, X-CLARITY, and EZ Clear.
- https://cdn.elifesciences.org/articles/77419/elife-77419-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Comparison of EZ Clear to solvent- and aqueous-based clearing methods.
(A) Schematic of required steps and time for tissue clearing with X-CLARITY, Fast 3D, 3DISCO, and EZ Clear. (B) Representative images of brains cleared by X-CLARITY, Fast 3D, 3DISCO, and EZ Clear …
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Figure 1—figure supplement 1—source data 1
Comparison of sample size changes of EZ Clear to solvent- and aqueous-based clearing methods before, during, and after clearing.
- https://cdn.elifesciences.org/articles/77419/elife-77419-fig1-figsupp1-data1-v1.xlsx
Figure 2
High refractive index imaging buffer EZ View achieves deeper imaging depth and maintains fluorescence stability.
(A) Refractive index (RI) of 80% Nycodenz increases linearly with increasing concentrations of urea (n=3). (B–D) Comparison of transparency of adult mouse brains processed with EZ Clear lipid …
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Figure 2—source data 1
Measurements for refractive index changes in response to different concentrations of Nycodenz and urea, quantitative comparison of fluorescence intensity of lectin-649 at different imaging depths between brains equalibrated in EZ View and RIMS, and fluorescence intensity measurement of lectin-649 after storing samples in EZ View for 14, 17, 43, and 70 days.
- https://cdn.elifesciences.org/articles/77419/elife-77419-fig2-data1-v1.xlsx
Figure 3
Quantitative comparison of fluorescence intensity and contrast between samples processed by EZ Clear and Fast 3D.
(A–F) Lightsheet imaging of a mouse brain perfused with Evans blue dye and cleared by EZ Clear (right hemisphere, A–C) and Fast 3D (left hemisphere, D–F) shows that tissue processed with EZ Clear …
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Figure 3—source data 1
Measurements and comparisons of fluorescent intensity and contrast of lectin-649 between EZ Clear and Fast 3D cleared mouse brains and organs.
- https://cdn.elifesciences.org/articles/77419/elife-77419-fig3-data1-v1.xlsx
Figure 4
EZ Clear processed samples are compatible with wholemount immunofluorescent staining.
(A) Six-step, standard wholemount immunofluorescent staining procedure for EZ Clear treated mouse brains. (B) Whole organ immunostaining of EZ Clear processed mouse brains using iDISCO or standard …
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Figure 4—source data 1
Measurements To-Pro 3 penetrance across the lateral axis between iDISCO and standard IHC processed samples.
Quantitative comparison of mean fluorescence intensity of To-Pro 3 and αSMA-Cy3 at different imaging depths (dorsal to ventral) between iDISCO and standard IHC processed samples.
- https://cdn.elifesciences.org/articles/77419/elife-77419-fig4-data1-v1.xlsx
Figure 5 with 1 supplement
EZ Clear processed and imaged samples can be further processed for downstream cryosection, histology, and immunofluorescence staining.
(A) Volume rendering of a wholemount lightsheet fluorescence microscopy (LSFM) imaged, EZ Clear processed postnatal day 104 (P104) glioblastoma multiforme (GBM) mouse brain with GFP+ tumor cells and …
Figure 5—figure supplement 1
EZ Clear processed and imaged samples processed for cryosectioning and hematoxylin and eosin (H&E) staining.
(A) Comparison of H&E staining of sections from brains that were either fixed in 4% paraformaldehyde (PFA) or processed with EZ Clear and imaged by lightsheet fluorescence microscopy (LSFM) at the …
Figure 6
EZ Clear processed and imaged samples are compatible with cryosectioning and immunofluorescent staining.
(A) Six-step immunostaining procedure for processing tissues after EZ Clearing and imaging. (B) Cryosections in the coronal plane from an EZ Clear processed and imaged postnatal day 104 (P104) …
Figure 7
Preparing a cleared mouse brain for imaging on the Zeiss Lightsheet Z.1 platform.
(A) A custom-designed sample holder with a magnetic nut. (B) A cleared brain sample was attached to the head of the plastic screw using superglue. (C) A 5 mL syringe is used as a casting mold for …
Videos
Video 1
Wholemount EZ Clear processed Thy1-EGFP adult mouse brain imaged by lightsheet fluorescent microscopy.
Video 2
Comparison of brain hemispheres following perfusion with Evans blue and clearing with either EZ Clear (right hemisphere) or Fast 3D (left hemisphere).
Video 3
Postnatal day 104 glioma brain perfused with DyLight 649-conjugated Lycopersicon esculentum (tomato) lectin (lectin-649).
Video 4
Three-dimensional (3D) volume rendering of a confocal stack from a glioblastoma multiforme (GBM) brain following cryosectioning and immunostained with GFAP (white) and Hoechst (blue) after EZ Clearing and lightsheet fluorescence microscopy (LFSM) imaging of the tumor (green) and blood vessels (red).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Lycopersicon esculentum (Tomato) Lectin (LEL, TL), DyLight 649 | Vector Laboratories | Cat #: DL-1178–1 | |
| Antibody | Rat Monoclonal Anti-Mouse CD31 | BD Biosciences | Cat #: 550274 | IF (1:200) |
| Antibody | Mouse Monoclonal Anti-actin, alpha-smooth muscle – Cy3 | Millipore-Sigma | Cat #: C6198 | IF (1:200) |
| Antibody | Rat Monoclonal Anti-GFAP | Invitrogen | Cat #: 13-0300 | IF (1:200) |
| Antibody | Rabbit Polyclonal Anti-beta III Tubulin | Abcam | Cat #: ab18207 | IF (1:200) |
| Antibody | Donkey Anti-Rat Alexa Fluor 568 | Abcam | Cat #: Ab175475 | IF (1:500) |
| Antibody | Donkey Anti-Rabbit Alexa Fluor 488 | Invitrogen | Cat #: A21206 | IF (1:500) |
| Antibody | Donkey Anti-Rabbit Alexa Fluor 568 | Invitrogen | Cat #: A10042 | IF (1:500) |
| Chemical compound, drug | Evans blue | Millipore-Sigma | Cat#: E2119 | |
| Chemical compound, drug | Histodenz | Millipore-Sigma | Cat#: D2158-100g | |
| Chemical compound, drug | Hoechst 33342 | Millipore-Sigma | Cat#: 14533 | 10 mg/mL stock at 1:1000 dilution |
| Chemical compound, drug | Diatrizoic acid | Millipore-Sigma | Cat#: D9268-1g | |
| Chemical compound, drug | Benzyl ether | Millipore-Sigma | Cat#: 108014 | |
| Chemical compound, drug | N-methyl-D-glucamine | Millipore-Sigma | Cat#: M2004-100g | |
| Chemical compound, drug | Triethylamine | Millipore-Sigma | Cat#: T0886 | |
| Chemical compound, drug | Tetrahydrofuran | Millipore-Sigma | Cat#: 186562 | |
| Chemical compound, drug | Nycodenz | Accurate Chemical & Scientific | Cat#: 100334–594 | |
| Chemical compound, drug | D-Sorbitol | Millipore-Sigma | Cat#: S1876 | |
| Chemical compound, drug | Urea | Millipore-Sigma | Cat#: U5378 | |
| Chemical compound, drug | Sodium azide | Millipore-Sigma | Cat#: S2002 | |
| Chemical compound, drug | VWR Life Science Agarose I | VWR | Cat#: 0710 | |
| Chemical compound, drug | Triton X-100 | Thermo Fisher Scientific | Cat#: BP-151 | |
| Chemical compound, drug | Donkey serum | Millipore-Sigma | Cat#: D9663 | |
| Chemical compound, drug | Na2HPO4 | Acros Organics | Cat#: 448140010 | |
| Chemical compound, drug | NaH2PO4 | Millipore-Sigma | Cat#: S0751 | |
| Chemical compound, drug | PBS, ×1 solution, pH 7.4, | Thermo Fisher Scientific | Cat#: BP24384 | |
| Chemical compound, drug | Paraformaldehyde | Millipore-Sigma | Cat#: P6148 | |
| Chemical compound, drug | Modified Harris Hematoxylin Solution | Millipore-Sigma | Cat#: HHS32-1L | |
| Chemical compound, drug | Eosin Y Phloxine B Solution | EMS | Cat#: 26051–21 | |
| Chemical compound, drug | Histoclear II | EMS | Cat#: 64111–04 | |
| Chemical compound, drug | DPX New | Millipore-Sigma | Cat#: 100579 | |
| Chemical compound, drug | Tissue-Tek OCT Compound | Sakura | Cat#: 4583 | |
| Software, algorithm | Imaris | Bitplane Inc | RRID:SCR_007370 | Ver. 8.5 |
| Software, algorithm | Vision4D | Arivis | RRID:SCR_018000 | Ver. 2.12 and 3.5 |
| Software, algorithm | Zeiss Zen Blue | Zeiss | RRID:SCR_013672 | Ver. 2.6.76.00000 (LSM 880) Ver. 1.1.2.0 (AxioZoom.V16 and AxioObserver.Z1) |
| Software, algorithm | Zeiss Zen Black | Zeiss | RRID:SCR_018163 | Ver. 9.2.5.54 (Lightsheet Z.1) Ver. 14.0.23.201 (LSM 880) |
| Software, algorithm | Labscope Material | Zeiss | Ver. 2.8.4 | |
| Software, algorithm | ImageJ/Fiji | NIH | RRID:SCR_002285 | Ver. 1.53c |
| Software, algorithm | Prism | GraphPad | RRID:SCR_002798 | Ver. 9.2.0 |
| Other | TO-PRO–3 Iodide (642/661) | Thermo Fisher Scientific | Cat#: T3605 | IF (1:1000) Nuclear counterstain |
| Other | Plastic Binding Head Slotted Screws, Off-White, 4–40 Thread Size, 1" Long | McMaster-Carr | Cat#: 94690A724 | Sample holder parts for lightsheet imaging |
| Other | Nylon Plastic Washer for Number 4 Screw Size, 0.112" ID, 0.206" OD | McMaster-Carr | Cat#: 90295A340 | Sample holder parts for lightsheet imaging |
| Other | Nylon Hex Nut, 4–40 Thread Size | McMaster-Carr | Cat#: 94812 200 | Sample holder parts for lightsheet imaging |
