Jun 29, 2026
Immunolabeling and Tissue Clearing for Calcified Tissues
- Zhangfan Ding1,2,
- Hanyu Liu1,
- Anjali Kusumbe1
- 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
- KUSUMBE LAB
Protocol Citation: Zhangfan Ding, Hanyu Liu, Anjali Kusumbe 2026. Immunolabeling and Tissue Clearing for Calcified Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.261geq45dg47/v1
Manuscript citation:
Biswas L, Chen J, De Angelis J, Singh A, Owen-Woods C, Ding Z, Pujol JM, Kumar N, Zeng F, Ramasamy SK, Kusumbe AP. Lymphatic vessels in bone support regeneration after injury. Cell. 2023 Jan 19;186(2):382-397.e24. doi: 10.1016/j.cell.2022.12.031.
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
Created: June 25, 2026
Last Modified: June 29, 2026
Protocol Integer ID: 319854
Keywords: Bone, Vasculature, Tissue clearing, imaging of intact skeletal tissue, tissue clearing for calcified tissue, sheet imaging of intact skeletal element, complex skeletal tissue architecture, calcified tissue, analysing complex skeletal tissue architecture, tissue clearing, prior experience in tissue clearing, standard tissue processing, mineralised tissue, bone microarchitecture, intact skeletal tissue, clearing methods for intact skeletal element, experience with standard tissue processing, imaging, bone marrow organization, resolution 3d imaging, encoded fluorescent reporter, sheet imaging, tissue, existing bone, skeletal development, bone collection, intact skeletal element, immunostaining technique, immunolabeling
Funders Acknowledgements:
European Research Council
Grant ID: StG: metaNiche, 805201
Ministry of Education (MOE) Singapore: Academic Research Funds
Grant ID: #024983-00001
Ministry of Education (MOE) Singapore: Academic Research Funds
Grant ID: #025277-00026
Disclaimer
The authors declare that they have no competing financial interests.
Abstract
High-resolution 3D imaging is an important strategy for visualising and analysing complex skeletal tissue architecture and the bone marrow microenvironment1–5,11. However, multicolour immunolabeling and imaging of intact skeletal tissues remain technologically challenging.1,5,8,10,11 . Current immunolabeling and tissue-clearing methods for intact skeletal elements often require prolonged processing times, extensive optimisation, limited antibody penetration into densely mineralised tissues, or rely on genetically encoded fluorescent reporters for specific applications1,5–11. Here, we describe a protocol for efficient clearing and immunolabeling of intact calcified tissues that enables ultrafast, single-cell-resolution, quantitative three-dimensional light-sheet imaging of intact skeletal elements and teeth.
The entire protocol, from bone collection to tissue clearing, takes approximately 4 days to complete, representing a substantial reduction in processing time compared with existing bone-clearing methods1,5,8,10,11. This protocol is broadly applicable to the visualisation of bone microarchitecture, bone marrow organization, vascular and neural network mapping, and the study of signalling molecules involved in skeletal development, regeneration, and ageing2–5,7,8,10,11. The protocol requires experience with standard tissue processing and immunostaining techniques, whereas prior experience in tissue clearing and light-sheet imaging is beneficial but not essential.
Attachments
Guidelines
- This protocol has been optimised for murine calcified tissues, including long bones, vertebrae, calvaria, mandibles, and teeth.
- A key aspect of our protocol is the addition of a collagenase digestion step after fixation and decalcification. This step enhances antibody penetration, resulting in deep, comprehensive staining throughout immunostained bones and other calcified tissues.
- Processing times for decalcification, collagenase digestion, and antibody incubation may require optimisation depending on tissue size, age, mineral density, and antibody penetration.
- Freshly prepared reagents, particularly collagenase solution, bleaching solution, and ECi-based clearing solution, should be used to ensure optimal performance.
- Samples should remain completely submerged throughout all incubation steps to ensure uniform reagent penetration and reproducible staining.
- Light exposure should be minimised after fluorescent antibody incubation to preserve fluorescence.
Materials
PBS solution (1×)
Fixation solution: 4% (wt/vol) PFA and 0.05% (vol/vol) glutaraldehyde in PBS. Divide the solution into aliquots and store them at -20 °C for up to 4 weeks.
Decalcification solution: 0.5 M EDTA in ddH2O (pH 7.4). Store it at 4 °C for 6 months.
Bleaching solution: 1.5% (vol/vol) H2O2 and 5% (vol/vol) DMSO in ethanol
Light-sheet buffer 1 (LSB1) : 25% (wt/vol) urea, 15% (vol/vol) glycerol, 7% (vol/vol) Triton X-100, and 10% (vol/vol) DMSO in ddH2O . Keep the buffer at 4 °C. Make up fresh each time and dispose of any unused solution properly.
Light-sheet buffer 2 (LSB2): 0.2% (wt/vol) collagenase A in PBS. It should be prepared freshly each time.
Blocking solution: 10% (vol/vol) DS, 10% (vol/vol) DMSO, and 0.5% (vol/vol) Triton X-100 in PBS. Prepare fresh each time.
Antibody dilution buffer: 2% (vol/vol) DS, 10% (vol/vol) DMSO, and 0.5% (vol/vol) Triton X-100 in PBS.
Light-sheet buffer 3 (LSB3): 2% (vol/vol) DS and 0.5% (vol/vol) Triton X-100 in PBS. Store it at 4 °C for up to 1 week.
Light-sheet buffer 4 (LSB4): 0.5% (vol/vol) Triton X-100 in PBS. Store it at 4 °C for up to 1 week.
Clearing medium: 80% (vol/vol) ECi and 20% (vol/vol) PEG
| Reagent | Supplier | Catalog number | |
| Phosphate-buffered saline (10×, PBS) | VWR | 437117K | |
| Paraformaldehyde (PFA) | Sigma-Aldrich | P6148 | |
| Glutaraldehyde | Sigma-Aldrich | 340855 | |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | E6758 | |
| Ethanol | Sigma-Aldrich | 1070172511 | |
| Hydrogen peroxide (H₂O₂) | Sigma-Aldrich | H1009 | |
| Triton X-100 | Sigma-Aldrich | T8787 | |
| Urea | VWR | 28876.367 | |
| Glycerol | VWR | 24388.260 | |
| Collagenase A | Merck | 10103578001 | |
| Fetal bovine serum (FBS) | Sigma-Aldrich | F7524 | |
| Donkey serum (DS) | Abcam | ab7475 | |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D5879 | |
| Methanol | Thermo Fisher Scientific | M/4058/17 | |
| Ethyl cinnamate (ECi) | Sigma-Aldrich | 112372 | |
| Polyethylene glycol (PEG) | Sigma-Aldrich | 447943 | |
| DAPI | Sigma-Aldrich | D9542 | |
| Superglue | No-Nonsense | 26780 |
| Item | Supplier | Catalog number | |
| 15 mL conical tube | Corning | 352096 | |
| 50 mL conical tube | Corning | 352070 | |
| Pipette (P10) | Gilson Pipetman | F144802 | |
| Pipette (P20) | Gilson Pipetman | F123600 | |
| Pipette (P200) | Gilson Pipetman | F123601 | |
| Pipette (P1000) | Gilson Pipetman | F123602 | |
| Pipette tips (P10) | Eppendorf | 0030000811 | |
| Pipette tips (P20) | Eppendorf | 0030000854 | |
| Pipette tips (P200) | Eppendorf | 0030000870 | |
| Pipette tips (P1000) | Eppendorf | 0030000927 | |
| 5 mL serological pipette | Corning | 4478 | |
| 10 mL serological pipette | Falcon | 357551 | |
| Serological pipette controller | Thermo Fisher Scientific | 10402822 | |
| Fine-tipped tweezers | Merck | T4287-1EA |
| Equipment | Supplier | Catalog number | |
| Rocking platform | VWR | 444-016 | |
| Water bath | Stuart | SBS40 |
Troubleshooting
Problem
Suboptimal decalcification effect or uneven decalcification
Solution
Fill the decalcifying solution to near the capacity of the tube and ensure that all regions of bone tissue are adequately immersed in the liquid during rotation
Do not use too small a tube during decalcification, as it may lead to incomplete decalcification.
Thoroughly stir for an adequate duration when preparing the EDTA solution
Adjust the pH of the decalcifying solution to 7.4
Problem
Suboptimal bleaching effect
Solution
Store H2O2 away from light and at 4 °C, as it degrades over time, resulting in a reduction in solution concentration
Dehydrate the sample strictly following the requirements of solution concentration gradients and time intervals
Problem
Weak fluorescence staining/no signals
Solution
After decalcification, make sure to thoroughly wash away EDTA based on the step requirements
Strictly control the bleaching time within 2 hours
Strictly adhere to the formulation of the permeabilization solution and incubation time
Adjust the antibody concentration based on the recommended dilution ratio
Avoid excessive dehydration and conduct dehydration at 4 °C to minimize the influences of ethanol and methanol on samples
Problem
Noisy and nonspecific fluorescent particles
Solution
Carefully follow the required washing steps for both primary and secondary antibodies
Problem
Poor sample transparency
Solution
Prolong the incubation time of the sample in the clearing medium
Problem
High background staining
Solution
Reduce the antibody concentration
Safety warnings
Paraformaldehyde (PFA), glutaraldehyde, methanol, Triton X-100, DMSO, and DAPI are hazardous chemicals. Handle all reagents in accordance with institutional safety guidelines using appropriate personal protective equipment (PPE). Prepare volatile or toxic reagents in a certified chemical fume hood and avoid inhalation, ingestion, and direct contact with the skin, eyes, and mucous membranes.
Ethics statement
All animal experiments should be approved by the appropriate institutional animal ethics committee and performed in accordance with local regulations and national guidelines for the care and use of laboratory animals.
Before start
Prepare all reagents and buffers before tissue collection. Freshly prepared collagenase solution, bleaching solution, antibody dilution buffer, and ECi clearing solution should be used. Pre-cool solutions that require 4°C storage and ensure that all samples can be fully submerged throughout the procedure. Prepare all required equipment, including a rocker, water bath, and 50 mL conical tubes, before starting.
Collection of samples
Cull 8-12 week-old mice (C57BL/6J mice) by cervical dislocation to isolate the bones. Collect all the bones carefully and wash them with PBS.
Bone fixation
2h 45m
Wash the freshly collected bones three times with PBS and immerse the cleaned bones immediately into the ice-cold fixation solution for 02:30:00
2h 30m
Wash all the samples 3 times with PBS at Room temperature for 00:05:00 each time.
Note
Ensure thorough washing to remove fixative agents.
15m
Decalcification
1d 0h 15m
Submerge bone tissue in 0.5 Molarity (M) EDTA solution (7.4 ) and incubate at 4 °C under constant rotation.
Note
Young mice for 24 hours and from aged mice for 48 hours.
1d
Wash all the samples three times with PBS on a rocker.
Each wash should last for 00:05:00 at 4 °C
15m
Dehydration
2h
Prepare the ethanol gradient in PBS. Immerse all the bones successively in an increasing gradient of 50%, 80%, 100% ethanol for dehydration. Incubate the samples in each solution at 4 °C for 00:30:00
2h
Bleaching
2h
Immerse dehydrated bone samples in bleach solution for 02:00:00 at Room temperature under gentle rotation.
Note
Avoid aqueous bleaching solutions, as they may cause rapid rehydration and tissue swelling.
2h
Rehydration
2h
Reverse the ethanol gradient to rehydrate the bones. Immerse all the samples with a decreasing gradient of ethanol concentrations at 4 °C . Wash all the samples three times for 00:20:00 each time.
2h
Antigen retrieval and permeabilization
5h
Prepare the ice-cold LSB1 and store the buffer at 4 °C until use.
Incubate the bone samples in LSB1 at 4 °C for 05:00:00
5h
Enzymatic digestion
40m
Incubate samples in freshly prepared LSB2 solution at 37 °C for 00:30:00 under constant shaking.
30m
Wash samples twice for 00:05:00 each in PBS at 37 °C on a rocker to stop digestion.
10m
Sample blocking
20m
Incubate the bone samples in blocking solution for 00:20:00 at 37 °C on a shaker.
Note
This step helps to prevent non- specific binding of antibodies and enhances signal specificity.
20m
Primary antibody incubation
14h
Incubate all samples with primary antibodies in antibody solution for 14:00:00 at 37 °C in a water bath with constant shaking at 70 rpm
Note
Recommended antibody concentrations for dilution in antibody dilution buffer are listed below.
| A | B | C | D | E | F | |
| SN. | Primary antibodies | Source | Host species | Identifier/RRID | Dilution | |
| 1 | LYVE1 | Abcam | Rabbit polyclonal IgG | CAT#ab14917; RRID: AB_301509 | 1:200 | |
| 2 | CD102 | BD Pharmingen | Rat Lewis IgG2a, κ | CAT#553326; RRID: AB_394784 | 1:300 | |
| 3 | FSP1 | Millipore | Rabbit polyclonal IgG | CAT#07-2274; RRID: AB_10807552 | 1:200 | |
| 4 | Endomucin | Santa Cruz Biotechnology | Rat monoclonal IgG2a | CAT#sc-65495; RRID: AB_2100037 | 1:300 | |
| 5 | GFP | ThermoFisher Scientific | Rabbit polyclonal IgG | CAT#A-21311; RRID: AB_221477 | 1:300 | |
| 6 | ⍺-SMA | Sigma-Aldrich | Mouse monoclonal IgG2a | CAT#C6198; RRID: AB_476856 | 1:300 | |
| 7 | Fibronectin | Abcam | Rabbit polyclonal IgG | CAT#ab2413; RRID: AB_2262874 | 1:300 | |
| 8 | HSPG2 | Sigma-Aldrich | Rat monoclonal IgG2a, κ | CAT#MAB1948P | 1:200 | |
| 9 | Laminin | Abcam | Rabbit polyclonal IgG | CAT#ab11575 | 1:200 | |
| 10 | Podocalyxin | B&D Systems | Goat polyclonal IgG | CAT#AF1556; RRID: AB_354858 | 1:200 | |
| 11 | Perilipin | Cell signaling | Rabbit monoclonal | CAT#9349; RRID: AB_10829911 | 1:300 | |
| 12 | Collagen I | Sigma-Aldrich | Rabbit polyclonal IgG | CAT#AB765P; RRID: AB_92259 | 1:300 | |
| 13 | CD31 | BD Pharmingen | Rat monoclonal Lewis IgG2a, κ | CAT#553370; RRID: AB_443021 | 1:100 | |
| 14 | CD31 | R&D Systems | Goat polyclonal IgG | CAT#AF3628; RRID: AB_2161028 | 1:100 | |
| 15 | Osteopontin | R&D Systems | Goat polyclonal IgG | CAT#AF808; RRID: AB_2194992 | 1:300 | |
| 16 | Osterix | Santa Cruz Biotechnology | Mouse monoclonal IgG1, κ | CAT#sc-393325; RRID: AB_2895257 | 1:200 | |
| 17 | Osteocalcin | Abcam | Rabbit polyclonal IgG | CAT#ab93876; RRID: AB_10675660 | 1:200 | |
| 18 | Osteocalcin | ThermoFisher Scientific | Rabbit polyclonal IgG | CAT# PA5-78870; RRID: AB_2745986 | 1:200 |
14h
Washing after antibody incubation
3h
Wash bone samples with LSB3 in a 37 °C water bath for 03:00:00 with gentle shaking at 70 rpm
3h
Secondary antibody incubation
6h
Incubate samples with secondary antibodies for 06:00:00 at 37 °C in a water bath with gentle shaking at 70 rpm
Note
Incubate the samples with DAPI (1:500) for an additional 1-2 hours. The recommended secondary antibodies and their dilution factors are listed below.
| SN. | Secondary antibodies | |||||
| 1 | Goat anti-Rabbit IgG (H + L) Alexa Fluor 546 | ThermoFisher Scientific | Goat polyclonal IgG | CAT#A-11035; RRID: AB_2534093 | 1:500 | |
| 2 | Donkey anti-Goat IgG (H + L) Alexa Fluor 488 | ThermoFisher Scientific | Donkey polyclonal IgG | CAT#A-11055; RRID: AB_2534102 | 1:500 | |
| 3 | Donkey anti-Goat IgG (H + L) Alexa Fluor 647 | ThermoFisher Scientific | Donkey polyclonal IgG | CAT#A-21447; RRID: AB_2535864 | 1:500 | |
| 4 | Donkey anti-Goat IgG (H + L) Alexa Fluor 546 | ThermoFisher Scientific | Donkey polyclonal IgG | CAT#A-11056; RRID: AB_2534103 | 1:500 | |
| 5 | Donkey anti-Rat IgG (H + L) Alexa Fluor 488 | ThermoFisher Scientific | Donkey polyclonal IgG | CAT#A-21208; RRID: AB_2535794 | 1:500 | |
| 6 | Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 | ThermoFisher Scientific | Donkey polyclonal IgG | CAT#A-21206; RRID: AB_2535792 | 1:500 | |
| 7 | Donkey anti-Rabbit IgG (H + L) Alexa Fluor 647 | ThermoFisher Scientific | Donkey polyclonal IgG | CAT#A-31573; RRID: AB_2536183 | 1:500 | |
| 8 | Donkey anti-Rat IgG H + L (Alexa Fluor 594) | ThermoFisher Scientific | Donkey polyclonal IgG | CAT#A-21209; RRID: AB_2535795 | 1:500 | |
| 9 | Streptavidin Alexa Fluor 647 conjugate | ThermoFisher Scientific | Streptavidin | CAT#S21374; RRID: AB_2336066 | 1:500 |
6h
Washing
3h
Wash bone samples with LSB3 in a 37 °C water bath for 03:00:00 under gentle shaking at 70 rpm
3h
Dehydration
2h 30m
Dehydrate all the samples by immersing them in an increasing gradient of ethanol (30%, 50%, and 80%) for 00:30:00 under gently rotating at 4 °C
1h 30m
Immerse the samples in 100% methanol for 01:00:00
Note
Change methanol every 20 minutes.
1h
Tissue clearing
30m
Remove the methanol completely and rinse the samples twice with 5 mL ECi each.
Clear the tissue samples by immersing them in the clearing solution and incubate at Room temperature for 00:30:00 under gentle rotation.
Note
Ensure that the tissues remain fully submerged throughout the clearing process.
30m
Protocol references
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Acknowledgements
A.P.K is supported by European Research Council (StG: metaNiche, 805201), Ministry of Education (MOE) Singapore: Academic Research Funds (#024983-00001, #025277-00026).