Jun 25, 2026
Immuolabelling and Tissue Clearing for Large Tissues and Organs
- Hanyu Liu1,
- Zhangfan Ding1,2,
- Anjali Kusumbe1,3
- 1Tissue and Tumor Microenvironments Lab, Cancer Discovery and Regenerative Medicine Program, Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 636921, Singapore;
- 2State Key Laboratory of Oral Diseases, National Center for Stomatology, National Clinical Research Center for Oral Diseases, Department of Head and Neck Oncology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China;
- 3Multidisciplinary Institute of Ageing (MIA-Portugal), University of Coimbra, 3004-504 Coimbra, Portugal
- KUSUMBE LAB
Protocol Citation: Hanyu Liu, Zhangfan Ding, Anjali Kusumbe 2026. Immuolabelling and Tissue Clearing for Large Tissues and Organs. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnp8nmv5d/v1
Manuscript citation:
Ding Z, Liu H, Chen J and Kusumbe AP. Protocol for ultrafast immunolabeling and 3D imaging of whole organs and large tissues. STAR Protocols, 2026; 7 DOI: 10.1016/j.xpro.2026.104623
Chen J, Ding Z, Biswas L, De Angelis J, Chatzis A, Kusumbe AP. Rapid 3D Immunolabeling and Light Sheet Microscopy for Quantitative Analysis of Intact Tissues. Comput Struct Biotechnol J. 2026 May 21;35(1):0121. doi: 10.34133/csbj.0121.
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: June 19, 2026
Last Modified: June 25, 2026
Protocol Integer ID: 319427
Keywords: Tissue clearing, Cell biology, Biotechnology and bioengineering, tissue clearing for large tissue, tissue immunolabelling, tissue clearing, human tissues for 3d imaging, organ imaging, whole soft tissue organ, molecular insights into tissue, human tissue, based tissue digestion, large tissue, 3d imaging, tissue digestion, sheet microscopy, tumour microenvironment, tissue, optical clearing, procedures for bleaching, immuolabelling, antigen retrieval, bleaching, collagenase, organ
Funders Acknowledgements:
Ministry of Education (MOE)
Grant ID: #024983-00001
Singapore: Academic Research Funds
Grant ID: #025277-00026
European Research Council
Grant ID: 805201
European Union’s Horizon 2020
Grant ID: no 857524
Disclaimer
The authors declare no competing interests.
Abstract
High-resolution whole-organ imaging provides cellular and molecular insights into tissue and tumour microenvironment. This protocol is able to process whole soft tissue organs and human tissues for 3D imaging within 2 to 2.5 days. We describe the procedures for bleaching, antigen retrieval, collagenase-based tissue digestion, whole-tissue immunolabelling, dehydration, and optical clearing for light-sheet microscopy.
Guidelines
This protocol is optimised for soft tissues. Processing times, antibody concentrations, and incubation durations may require optimisation depending on tissue size, tissue composition, and target antigen abundance. Users should perform pilot experiments when applying the protocol to new tissues, species, or antibody combinations.
Materials
Buffers:
Fixation buffer: 4% PFA and 0.05% glutaraldehyde in 1X PBS.
Bleaching buffer: 1.5% of 30% H2O2 in ethanol
Antigen retrieval solution: 25% (w/v) urea+ 15% (v/v) glycerol+ 7% (v/v) Triton X-100 in ddH2O.
Digestion buffer: 0.2% Collagenase A in 1X PBS.
Washing buffer 1 : 2% FBS in 1X PBS
Blocking buffer: 10% DS+ 10% DMSO + 0.5% Triton X-100 in 1X PBS
Antibody buffer: 2% DS+ 10%DMSO+ 0.5% Triton X-100 in PBS; antibodies 1:500
Washing buffer 2 : 2% DS+ 0.5% Triton X-100 in PBS
Clearing buffer : 80% ECi + 20% PEGM
| A | B | C | |
| Chemicals, peptides, and recombinant proteins | |||
| Paraformaldehyde (PFA) | Sigma-Aldrich | P6148 | |
| Collagenase A | Merk | 10103578001 | |
| Dimethyl sulfoxide | Sigma-Aldrich | D5879 | |
| Donkey serum | Abcam | ab7475 | |
| Ethyl cinnamate | Sigma-Aldrich | 112372 | |
| Evans Blue | Sigma-Aldrich | E2129 | |
| Fetal bovine serum | Sigma-Aldrich | F7524 | |
| Glutaraldehyde | Sigma-Aldrich | 340855 | |
| Glycerol | VWR | 24388.260 | |
| 30% H2O2 | Sigma-Aldrich | H1009 | |
| Ethanol | Sigma-Aldrich | 1070172511 | |
| Paraformaldehyde | Sigma-Aldrich | P6148 | |
| DMSO | Sigma-Aldrich | D5879 | |
| PBS | VWR | 437117 K | |
| poly(ethylene glycol) methyl ether methacrylate (PEGM) | Sigma-Aldrich | 447943 | |
| Tamoxifen | Sigma-Aldrich | T5648 | |
| Triton X-100 | Sigma-Aldrich | T8787 | |
| Urea | VWR | 28876.367 | |
| Antibodies | |||
| VEGFR2 | Abcam | ab11939 | |
| α-SMA | Sigma-Aldrich | C6198 | |
| Endoglin | R&D Systems | AF1320 | |
| CD8⍺ | Abcam | ab237368 | |
| LYVE1 | Abcam | ab14917 | |
| Endomucin | Santa Cruz Biotechnology | sc-65495 | |
| Donkey anti-Rat IgG Alexa Fluor 594 | Thermo Fisher Scientific | A21209 | |
| Donkey anti-Goat IgG Alexa Fluor 488 | Thermo Fisher Scientific | A11055 | |
| Donkey anti-Goat IgG Alexa Fluor 647 | Thermo Fisher Scientific | A21447 | |
| Donkey anti-Goat IgG Alexa Fluor 546 | Thermo Fisher Scientific | A-11056 | |
| Alexa Fluor 488 streptavidin conjugate | Thermo Fisher Scientific | S11223 | |
| Alexa Fluor 546 streptavidin conjugate | Thermo Fisher Scientific | S11225 | |
| Donkey anti-Rabbit IgG Alexa Fluor 488 | Thermo Fisher Scientific | A21206 | |
| Donkey anti-Rabbit IgG Alexa Fluor 647 | Thermo Fisher Scientific | A31573 | |
| Shaking water bath | |||
Troubleshooting
Problem
High background noise
Solution
Perform cardiac perfusion thoroughly before tissue collection, particularly for highly vascularized organs, to minimise residual blood and hemosiderin deposition.
Apply a bleaching step to reduce endogenous pigmentation and hematoma-derived background signals.
During dehydration, ensure samples are completely immersed in ethanol. Incubate tissues in each ethanol gradient for 30–40 minutes to achieve efficient dehydration and optimal clearing performance.
Problem
Strong signal at the tissue surface with weak internal labeling.
Solution
Extend the digestion/permeabilisation time to facilitate deeper antibody penetration. Longer primary antibody incubation and optimised detergent treatment may further improve staining uniformity throughout the tissue.
Problem
Weak antibody signals
Solution
Increase the antibody concentration or prolong antibody incubation time. Additional optimisation of tissue permeabilisation and staining conditions may further enhance signal intensity.
Problem
High antibody background signal
Solution
Ensure thorough washing between antibody incubation steps to remove unbound antibodies. Increase the number and duration of washes if needed
Problem
Bubbles inside the organs
Solution
Gently fill bubble-containing spaces with clearing solution using a fine needle while avoiding tissue damage.
Safety warnings
Paraformaldehyde, glutaraldehyde, hydrogen peroxide, ethanol, dimethyl sulfoxide (DMSO), and ethyl cinnamate (ECi) are hazardous chemicals and should be handled in a chemical fume hood with appropriate PPE in accordance with institutional safety guidelines.
Ethics statement
All animal studies were approved by the relevant institutional and local authorities and performed in accordance with applicable guidelines and regulations. Human samples were obtained under local ethics committee-approved protocols with informed consent from all participants.
Before start
Advanced three-dimensional imaging provides critical insights into complex biological processes during organ development, regeneration, repair, and disease1,2,3,4,5,6,7,8,9. However, the acquisition of high-content 3D datasets remains challenging because it often requires extensive sample preparation, prolonged processing times, large data storage capacity, and sophisticated analytical workflows10,11,12,13,14,15. In this protocol, we describe an ultrafast immunolabelling and solvent-based optical clearing method for intact organs.
This protocol has been specifically optimised for soft tissues and organs but can be readily adapted to other murine organs and selected human tissue samples provided suitable antibodies are available. Processing times may require adjustment according to tissue size, density, and antigen accessibility.
For highly vascularised organs, cardiac perfusion is recommended to minimise residual blood-derived pigments and improve staining quality. Additional bleaching steps may be required to enhance tissue transparency in samples containing abundant endogenous pigmentation. Larger tissue specimens may also require extended incubation times during immunolabelling and clearing to ensure adequate reagent penetration. Overall, this rapid and reproducible workflow enables high-resolution whole-organ imaging and facilitates the analysis of complex tissue architecture, vascular networks, and rare cellular populations.
Tissue collection and fixation
3h 10m
Freshly dissect the tissues and rinse them in PBS.
Note
- It is recommended to remove excess muscle and adipose tissue before fixation.
- Cardiac perfusion is recommended, especially for the organs with an abundant blood supply, such as the heart, spleen, and kidney.
Transfer samples into fixative solution at 4 °C 03:00:00 on a shaker
Note
The volume of fixative solution should be at least ten times that of the samples to ensure proper fixation.
3h
Wash samples 3 times with PBS on a shaker. Each wash lasted for 00:05:00 at Room temperature
Note
The fixed samples can be stored for up to 4 days at 4°C
10m
Bleaching
4h
Immerse samples in ethanol gradient of 50%,80% and 100% for 00:30:00 each for dehydration.
100% ethanol was changed twice, 00:30:00 apart.
1h
Immerse samples in bleaching buffer at 4 °C for up to 03:00:00
Note
Avoiding light
Safety information
Hydrogen peroxide (H₂O₂) in bleaching buffer is a hazardous oxidizing agent and should be handled in a chemical fume hood.
3h
Rehydration
30m
Immerse organs in an ethanol gradient of 100%, 80% and 50% for 00:30:00 each for rehydration.
30m
Antigen retrieval
18h
Incubate tissues with antigen retrieval buffer4 °C for 06:00:00
Note
The time of antigen retrieval depends on the sample size and tissue density. 6 h is generally sufficient for small mouse organs, whereas larger organs may require up to 12 h.
18h
Collagenase digestion
1h 5m
Incubate samples with the digestion solution at 37 °C 00:30:00 with constant shaking
Note
The time of digestion should be limited to 30 min to avoid damage to tissue integrity and ensure proper permeability of the samples.
1h
Washing samples twice with washing buffer 1 for 00:05:00 each
5m
Immunolabelling
1d 3h 5m
Incubate samples in blocking solution at 70 rpm, 37°C, 00:20:00 in a water bath
20m
Incubate samples with primary antibody solution 70 rpm, 37°C, 14:00:00 in the water bath
Note
Antibody concentrations may vary depending on tissue size, target antigen expression levels, antibody quality, and tissue permeability. As a result, staining conditions should be optimised for individual experiments. The antibody concentrations listed below according to our experience:
| ANTIBODY | Dilution | |
| VEGFR2 | 1:300 | |
| α-SMA | 1:200 | |
| Endoglin | 1:300 | |
| CD8⍺ | 1:300 | |
| LYVE1 | 1:200 | |
| Endomucin | 1:300 |
14h
Wash samples with washing buffer 2 for 03:00:00 with the same procedure above at 70 rpm, 37°C in the shaking water bath.
Note
The wash buffer was changed every 00:15:00 for the first hour and then every 00:30:00 for the remaining 2 hours.
3h 45m
Incubate samples in secondary antibody buffer for 06:00:00 at 37 °C in the shaking water bath rotating at 70 rpm.
Note
Antibody concentrations may vary depending on tissue size, target antigen expression levels, antibody quality, and tissue permeability. As a result, staining conditions should be optimised for individual experiments. The antibody concentrations listed below according to our experience:
| Donkey anti-Rat IgG Alexa Fluor 594 | 1:500 | |
| Donkey anti-Goat IgG Alexa Fluor 488 | 1:500 | |
| Donkey anti-Goat IgG Alexa Fluor 647 | 1:500 | |
| Donkey anti-Goat IgG Alexa Fluor 546 | 1:500 | |
| Alexa Fluor 488 streptavidin conjugate | 1:500 | |
| Alexa Fluor 546 streptavidin conjugate | 1:500 | |
| Donkey anti-Rabbit IgG Alexa Fluor 488 | 1:500 | |
| Donkey anti-Rabbit IgG Alexa Fluor 647 | 1:500 |
6h
Wash samples with washing buffer 2 for 03:00:00 with the same procedure above at 70 rpm, 37°C in the shaking water bath
3h
Dehydration
1h 30m
Immerse samples in a gradient of 30%, 50% and 80% ethanol for 00:30:00 each under gentle rotation Room temperature
30m
Immerse samples in 100% ethanol and replace the ethanol twice.
1h
Clearing
1h 40m
Completely remove ethanol
Rinse samples twice with ethyl cinnamate (ECi) for 00:10:00 each.
Safety information
ECi is an irritant and should be handled in the fume hood, and the appropriate PPE is required.
10m
Clear samples with 80% ECi and 20% poly(ethylene glycol) methyl ether methacrylate (PEGM) under gentle rotation at Room temperature
1h 30m
Protocol references
1. Kumar, N., Saraber, P., Ding, Z., and Kusumbe, A.P. (2021). Diversity of vascular niches in bones and joints during homeostasis, ageing, and diseases. Front. Immunol. 12, 798211. https://doi.org/10.3389/fimmu.2021.798211.
2. Shi, M.Y., Yao, Y., Wang, M., Yang, Q., Ding, L., Li, R., Li, Y., Huang, H., Yang, C.Y., Zhou, Z., et al. (2025). High-speed mapping of whole mouse peripheral nerves at subcellular resolution. Cell 188, 3897–3915.e20. https://doi.org/10.1016/j.cell.2025.06.011.
3. Ou, Z., Duh, Y.S., Rommelfanger, N.J., Keck, C.H.C., Jiang, S., Brinson, K., Jr., Zhao, S.,Schmidt, E.L., Wu, X., Yang, F., et al. (2024).
Achieving optical transparency in live animals with absorbing molecules. Science 385, eadm6869. https://doi.org/10.1126/science. adm6869.
4. Ding, Y., Ma, J., Langenbacher, A.D., Baek, K.I., Lee, J., Chang, C.C., Hsu, J.J., Kulkarni, R.P., Belperio, J., Shi, W., et al. (2018). Multiscale light-sheet for rapid imaging of cardiopulmonary system. JCI Insight 3, e121396. https://doi.org/10.1172/jci.insight.121396.
5. Udan, R.S., Piazza, V.G., Hsu, C.W., Hadjantonakis, A.K., and Dickinson, M.E. (2014). Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy. Development 141, 4406–4414. https://doi.org/10.1242/dev.111021.
6. Takebe, T., Sekine, K., Kimura, M., Yoshizawa, E., Ayano, S., Koido, M., Funayama, S., Nakanishi, N., Hisai, T., Kobayashi, T., et al. (2017). Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells. Cell Rep. 21, 2661–2670. https:// doi.org/10.1016/j.celrep.2017.11.005.
7. Chen, J., Sivan, U., Tan, S.L., Lippo, L., De Angelis, J., Labella, R., Singh, A., Chatzis, A., Cheuk, S., Medhghalchi, M., et al. (2021). High resolution 3D imaging uncovers organ-specific vascular control of tissue aging. Sci. Adv. 7, eabd7819. https://doi.org/10.1126/sciadv.abd7819.
8. Chen, J., Lippo, L., Labella, R., Tan, S.L., Marsden, B.D., Dustin, M.L., Ramasamy, S.K., and Kusumbe, A.P. (2021). Decreased blood vessel density and endothelial cell subset dynamics during ageing of the endocrine system. EMBO J. 40, e105242. https://doi.org/10.15252/embj.2020105242.
9. Stucker, S., De Angelis, J., and Kusumbe, A.P. (2021). Heterogeneity and dynamics of vasculature in the endocrine system during aging and disease. Front. Physiol. 12, 624928. https://doi.org/10.3389/fphys.2021.624928.
10. Yi, Y., Li, Y., Zhang, S., Men, Y., Wang, Y., Jing, D., Ding, J., Zhu, Q., Chen, Z., Chen, X., et al.
(2024). Mapping of individual sensory nerve axons from digits to spinal cord with the transparent embedding solvent system. Cell Res. 34, 124–139. https://doi.org/10.1038/s41422-023-00867-3.
11. Messal, H.A., Almagro, J., Zaw Thin, M.,Tedeschi, A., Ciccarelli, A., Blackie, L., Anderson, K.I., Miguel-Aliaga, I., van Rheenen, J., and Behrens, A. (2021). Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH. Nat. Protoc. 16, 239–262. https:// doi.org/10.1038/s41596-020-00414-z.
12. Sato, Y., Miyawaki, T., Ouchi, A., Noguchi, A., Yamaguchi, S., and Ikegaya, Y. (2019). Quick visualization of neurons in brain tissues using an optical clearing technique. Anat. Sci. Int. 94, 199–208. https://doi.org/10.1007/s12565-018-00473-z.
13. Mai, H., Luo, J., Hoeher, L., Al-Maskari, R., Horvath, I., Chen, Y., Kofler, F., Piraud, M., Paetzold, J.C., Modamio, J., et al. (2024). Whole-body cellular mapping in mouse using standard IgG antibodies. Nat. Biotechnol. 42, 617–627. https://doi.org/10.1038/s41587-023-01846-0.
14. Power, R.M., and Huisken, J. (2017). A guide to light-sheet fluorescence microscopy for multiscale imaging. Nat. Methods 14, 360–373. https://doi.org/10.1038/nmeth.4224.
15. Otomo, K., Omura, T., Nozawa, Y., Edwards, S.J., Sato, Y., Saito, Y., Yagishita, S., Uchida, H., Watakabe, Y., Naitou, K., et al. (2024). descSPIM: an affordable and easy-to-build light-sheet microscope optimized for tissue clearing techniques. Nat. Commun. 15, 4941. https://doi.org/10.1038/s41467-024-49131-1.
Acknowledgements
A.P.K. is supported by the Ministry of Education (MOE), Singapore: Academic Research Funds (#024983-00001 and #025277-00026), the European Research Council (StG: metaNiche, 805201), and the European Union’s Horizon 2020 (no 857524).