Mar 23, 2026
c-Clear 3-D Tissue Clearing Protocol
- Mairobys Socorro1,
- Janak Gaire2,
- Juliane Rolim de Lavor1,
- Yenisel Cruz-Almeida2,
- Robert Caudle2,
- Reese Smith1,
- Alan Watson1,
- Kyle Allen2,
- Alejandro Almarza1
- 1University of Pittsburgh;
- 2University of Florida
- RE-JOIN
Protocol Citation: Mairobys Socorro, Janak Gaire, Juliane Rolim de Lavor, Yenisel Cruz-Almeida, Robert Caudle, Reese Smith, Alan Watson, Kyle Allen, Alejandro Almarza 2026. c-Clear 3-D Tissue Clearing Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjex6ngk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 22, 2025
Last Modified: March 23, 2026
Protocol Integer ID: 223015
Keywords: Knee joint, TMJ, 3D, Tissue clearing, Rats, Sensory innervation, Immunolabeling, Light-sheet imaging, clearing of rat knee joint, existing tissue clearing tool, tissue clearing tools such as cubic, transparency of tissue, rat knee joint, clear protocol, human tissue, clearing, transparency, effective for rat, tissue
Funders Acknowledgements:
National Institute of Arthritis and Musculoskeletal and Skin Diseases
Grant ID: UC2AR082196
Abstract
c-Clear protocol was designed by combining features of existing tissue clearing tools such as CUBIC (Susaki et al, 2014) and 3DISCO (Ertürk et al., 2012) to enhance transparency of tissues compared to any specific protocol. Although, the protocol was designed for staining and clearing of rat knee joints, we have found it to be highly effective for rat temporomandibular joint (TMJ) and human tissue, including but not limited to knee fat pad.
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Materials
| Reagent | Supplier | Amount | Cat. No. | |
| Bovine serum albumin (BSA) | Sigma-Aldrich | 100g | A2153-100g | |
| Dibenzyl Ether (DBE) | Sigma-Aldrich | 1L | 33630-1L | |
| Dichloromethane (DCM) | Sigma-Aldrich | 1L | 270997-1L | |
| Hydrochloric acid (HCL) | Thermo Fisher | 500 mL | A144S-500 mL | |
| Immunocal | StatLab | 1 GAL | SKU #: 1414-1 GAL | |
| Normal Goat Serum (NGS) | Gibco | 100 mL | 16210064-100 mL | |
| Paraformaldehyde (PFA) | Thermo Fisher | 500g | 416780250-500g | |
| PBS, ×10 solution, pH 7.4 | Thermo Fisher | 4L | BP399-4-4L | |
| Quadrol | Sigma-Aldrich | 1L | 122262-1L | |
| Sodium Azide | Sigma-Aldrich | 25g | S2002- 25g | |
| Sodium Hydroxyde (NaOH) | Thermo Fisher | 1kg | CAS: 1310-73-2-1kg | |
| Tetrahydrofuran (THF) | Sigma-Aldrich | 2L | 186562-2L | |
| TritonX-100 | Thermo Fisher | 500 mL | BP151-500mL | |
| Tween 20 | Sigma-Aldrich | 250 mL | P1379- 250 mL | |
| Urea | Sigma-Aldrich | 1kg | U5378-1kg |
Reagents used to perform c-Clear 3D tissue-clearing
| Description | Supplier | Cat. No. | |
| 50 mL conical tubes (polypropylene) | Thermo Fisher | 14-432-22 | |
| Aluminium foil | MLS | - | |
| Distilled water | MLS | - | |
| Filter paper | Thermo Fisher | 09-801B | |
| Fish fryer | (Custom made) | - | |
| Micro-dissecting forceps | MLS | - | |
| Fume hood | MLS | - | |
| Incubator (37 degrees C) | MLS | - | |
| Incubator (RT) | MLS | - | |
| Surgical scalpel blades | MLS | - | |
| Surgical scissors | MLS | - | |
| Platform rotator | MLS | - | |
| Nutator | MLS | - |
Other materials. MLS: Major Laboratory Supplier in North America.
Troubleshooting
Safety warnings
Appropriate personal protective equipment (PPE), including gloves and a lab coat, must be worn at all times while performing this procedure. Tetrahydrofuran (THF) is a volatile and flammable solvent and must be handled exclusively within a certified chemical fume hood, including during reagent preparation. All chemical reagents should be properly disposed of as hazardous chemical waste in accordance with institutional safety guidelines.
Ethics statement
All animal procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (protocol #22112127) and conducted under the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Tissue Clearing
Prior to tissue clearing, animals (rats) should undergo transcardial perfusion to ensure proper fixation. Perfusion is first carried out with approximately 200 mL of cold 0.01 M phosphate-buffered saline (PBS, pH 7.4) to flush the blood and prevent clotting, immediately followed by an equal volume of cold 4% PFA (4% paraformaldehyde in 0.01 M PBS, pH 7.4) to fix the tissue.
30m
Note
All incubation steps are carried out under gentle agitation to ensure even reagent distribution (a nutator is ideal).
Fix tissues in 4% PFA for 24 hours at 4°C.
Note
Immerse in three volumes of 4% PFA, or at least enough to cover the tissue.
1d
Wash the tissues with 1X PBS to remove excess fixative. Repeat this step twice (intervals of 3–4 hours) and collect the waste in a chemical hazard container.
Note
Samples can be stored in 1X PBS + 0.02% sodium azide at 4°C until further processing.
8h
Place each sample in a 50 mL conical tube containing Immunocal (StatLab, SKU#:1414) and incubate at 4°C with gentle agitation on a rocker set at 70 revolutions per minute (RPM). Change the Immunocal solution every 24-48 hours until tissue is soft.
Note
The adult rat knee takes approximately ~ 7-9 days to decalcify.
1w
After decalcification, replace Immunocal with distilled water and wash tissues on a shaker for at least 30 min. Repeat this step.
1h
If whole sample is not desired, cut sample in half (bisected in the sagittal plane) or in quarters using a sharp razor blade and place each sample in a 50 ml conical tube with IHC buffer.
Note
Whole knee samples are immersed immediately in IHC buffer.
15m
Rinse sample(s) with IHC buffer for 1 hour at room temperature.
1h
Immerse tissues in 50% CUBIC R1 (dilute 100% CUBIC R1(stock) 1:1 with dH2O), incubate at 37°C for 24 hours.
1d
Replace with 50% CUBIC R1, incubate at 37°C, and replace every 24-28 hours.
4d
After 4 days, replace with 100% CUBIC R1, incubate at 37°C and change the solution every 24 hours.
Note
Expect tissue and solution to turn a green/brown color due to CUBIC R1 dissolving iron-based chromophores like heme. A good indication of when the tissue is ready for staining is when this color is removed after multiple changes of CUBIC R1. The tissue may not appear translucent or clear at this step.
The rate of tissue clearing depends of tissue thickness and temperature of incubation: Whole rat knees are generally complete within 7-9 days.
3d
Immediately, proceed to process samples for photobleaching and immunolabeling
Photobleaching (CUBIC-Cleared Tissues)
2d 7h
After clearing in 100% CUBIC R1, wash with IHC buffer 3x for 1 hour each at 37°C.
Note
Use a generous volume – at least 3x the volume of the tissue being cleared.
3h
Dehydrate samples using THF Series.
Safety information
All steps that involve manipulation of THF should be done under a fume hood.
Note
Each incubation step should use a volume of solution that is at least 3x greater than the volume of the tissue being cleared.
30% THF for 2-hours, gentle movement at room temperature (RT).
2h
50% THF for 2-hours, gentle movement at RT.
2h
70% THF for 2-hours, gentle movement at RT.
2h
90% THF for 2-hours, gentle movement at RT. Repeat with fresh 90% THF.
4h
100% THF for 2-hours, gentle movement at RT. Repeat with fresh 100% THF.
4h
Clear samples with Dibenzyl Ether (DBE) for 1 hour at RT.
Note
The tissue should noticeably begin to clear within 30 minutes of adding DBE.
1h
Change DBE at least once. Incubate until clearing appears to have completed (usually 24-28 hours).
1d
Place tissues in the fish fryer at desired wavelength (usually 488 for autofluorescence) for 8 hours (photobleaching step). Tissues should be immersed in DBE at all time.
Note
The duration of photobleaching should be adjusted based on the anticipated level of autofluorescence and the size of the tissue sample. Larger tissues or those with higher background fluorescence will require longer photobleaching to effectively reduce signal interference. Whole rat knees are photobleached for 8h.
8h
Rehydrate samples through stepwise incubation in a descending series of THF concentrations.
Safety information
All steps that involve manipulation of THF should be done under a fume hood.
Note
Each incubation step should use a volume of solution that is at least 3x greater than the volume of the tissue being cleared.
100% THF for 1-hour, gentle movement at RT.
1h
90% THF for 1-hour, gentle movement at RT.
1h
70% THF for 1-hour, gentle movement at RT.
1h
50% THF for 1-hour, gentle movement at RT.
1h
30% THF for 1-hour, gentle movement at RT.
1h
Immediately, proceed to the immunolabeling step.
Immunolabeling
2w 3d 5h
Wash samples with IHC buffer 3x for 1 hour each at 37°C.
3h
Block samples in freshly prepared blocking buffer (1% Bovine serum albumin, 4% normal goat serum, 0.2% Triton-X prepared in 1X PBS) at 4 °C on a shaker (use 3-4 mL per sample).Overnight
Note
The volume will depend on the size of the sample. Tissue samples must remain fully immersed in the blocking solution throughout the entire incubation step.
1d
Incubate samples in primary antibody diluted in staining buffer [4% normal goat serum, 0.2% Tween-20 in 1X PBS (PBST)] at 4 °C on a shaker for 7 days. Ensure that the samples are fully submerged in the buffer solution.
1w
Wash samples in PBST for a day. First, a quick rinse 1X PBS with 0.02% Tween followed by incubating samples in 8 mL of PBST at 4 °C on a shaker for 3-4 hours. Repeat this step twice.
Note
Samples can be left in PBST overnight at 4 °C on a shaker (preferred).
1d
Incubate the samples in secondary antibody in staining buffer at 4 °C on a shaker for 7 days. Here onwards, protect samples from light exposure.
1w
Wash samples in PBST for a day. First, a quick rinse with 4 mL of PBST followed by incubating samples in 8 mL of PBST at 4 °C on a shaker for 3-4 hours. Repeat this step twice.
Note
Samples can be left in PBST overnight at 4 °C on a shaker (preferred).
1d
Post-fix the tissue(s) in 4% PFA at room temperature for 1 hour.
Note
Fixation preserves labeling by creating crosslinks that stabilize the sample. Since clearing often involves prolonged imaging sessions, post-fixation is advantageous to retain signal. This is helpful when storing antibody-stained samples for an extended period.
1h
Wash the tissue for 1 hour in IHC buffer under continuous gentle agitation. After the first 30 minutes, replace the solution with fresh IHC buffer.
1h
Refractive index matching with DISCO
1d 20h
Dehydrate samples using THF Series.
Safety information
All steps that involve manipulation of THF should be done under a fume hood.
Note
Each incubation step should use a volume of solution that is at least 3x greater than the volume of the tissue being cleared.
30% THF for 2-hours, gentle movement at RT.
2h
50% THF for 2-hours, gentle movement at RT.
2h
70% THF for 2-hours, gentle movement at RT.
2h
90% THF for 2-hours, gentle movement at RT. Repeat with fresh 90% THF.
4h
100% THF for 2-hours, gentle movement at RT. Repeat with fresh 100% THF.
4h
Incubate in DCM (Dichloromethane) for 1 hour at RT.
1h
Incubate in DCM for 2 hours at RT. Repeat with fresh DCM.
4h
Clear with Dibenzyl Ether (DBE) for 1 hour. Room temperature
Note
The tissue should noticeably begin to clear within 30 minutes of adding DBE.
1h
Change DBE at least once. Incubate until clearing appears to have completed (usually 24-28 hours).
Note
Samples are stored in the dark to prevent light exposure. Room temperature
1d
Mount and image in DBE using a light-sheet fluorescence microscope (mesoSPIM). Smaller samples can also be imaged using a ribbon scanning confocal microscope.
Note
Samples are imaged following completion of tissue-clearing and immunolabeling protocols.
Preparation of Solutions
CUBIC R1 (500g; ~420ml): CUBIC R1 (100% stock) 50% CUBIC R1
Urea 25wt% 125g 62.5g
Quadrol 25wt% 125g 62.5g
TritonX-100 15wt% 70mL 35mL
dH2O 35wt% 175mL 175mL
--Quadrol= N,N,N′,N′-Tetrakis(2-hydroxypropyl)ethylenediamine
--Quadrol should be weighed directly into the urea solution rather than measured by volume (very viscous).
Preparation of CUBIC R1:
- Mix 125g of Urea and 175mL of H2O in a glass beaker.
- Stir on a hot plate over low heat or place in a water bath, up to 56 °C, until the urea dissolves.
- Add 125g of Quadrol (N,N,N′,N′-Tetrakis(2-Hydroxypropyl)ethylenediamine,
- Stir over low heat until the Quadrol dissolves.
- Add 70mL of TritonX-100.
- Remove from heat and stir until dissolved.
Note
Store the solution sealed at room-temperature. The shelf-life is approximately 1 month. When the solution takes on a strong ammonia smell, it has expired. If the temperature is too high when making the solution, the ammonia smell will be immediately present, and the solution should be discarded.
IHC Buffer (500mL):
0.1% TritonX-100 500uL
0.5% BSA 2.5g
0.02% Sodium Azide 0.1g
1X PBS 499mL
THF – dilution series (30%, 50%, 70%, 90%) (250mL)
30% 50% 70% 90%
75mL 125mL 175mL 225mL THF
175mL 125mL 75mL 25mL dH2O
8% Paraformaldehyde Solution (PFA)
- Add 24 grams of paraformaldehyde resin to 210 ml of 1X PBS
- Heat to 70°C (does not dissolve)
- Add 1M NaOH till the solution clears, (usually a couple of drops)
- Cool to room temperature
- Add 1X PBS to bring the total volume up to 300mL
- Use NaOH/HCl to attain a pH 7.4 (use pH strips)
- Filter (Fisherbrand‱ Qualitative Grade Plain Filter Paper Circles - P5 Grade)
- Store at 4°C protected from the dark (up to 3 months).
Note
Freshly prepared and properly pH-adjusted PFA should be used for perfusion and tissue fixation prior to clearing. We have found that using freshly prepared, pH-balanced PFA helps minimize autofluorescence. The PFA is made and stored at 8% for a maximum of 3 months at 4°C (protected from the light). 8% PFA is diluted to 4% with 1X PBS before use.
4% Paraformaldehyde Solution (PFA)
- Dilute 50ml of the 8% paraformaldehyde stock with 50 ml of 1X PBS.
- Keep the solution at 4°C and use the same day.
Acknowledgements
- We thank the Department of Cell Biology and Center for Biologic Imaging (CBI) at the University of Pittsburgh, for providing technical support to perform all 3D clearing and imaging experiments. This work was funded by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (UC2AR082196) and National Institutes of Health Office of the Director S10OD038197 to Alan M. Watson.