Sep 24, 2025
3D Visualization of Neural Innervation in Rat Knee Joint Using PEGASOS-based Tissue Clearing
- Janak Gaire1,
- Mairobys Socorro2,
- Alejandro Almarza2,
- Kyle Allen1
- 1University of Florida;
- 2University of Pittsburgh
- RE-JOIN
Protocol Citation: Janak Gaire, Mairobys Socorro, Alejandro Almarza, Kyle Allen 2025. 3D Visualization of Neural Innervation in Rat Knee Joint Using PEGASOS-based Tissue Clearing . protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw4947lmk/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 17, 2025
Last Modified: September 24, 2025
Protocol Integer ID: 222738
Keywords: Tissue clearing, Knee Joint, 3D microscopy, Joint Innervation, resolution mapping of joint innervation, 3d visualization of neural innervation, rat knee joint, joint innervation, visualizing neurofilament, nerves in intact joint, imaging nerve, nerve fiber, knee joint, osteoarthritis, nerve fibers in whole, neural innervation, based tissue clearing, tissue clearing method, critical role in osteoarthritis, rat knee, innervation, tissue clearing, intact joint, polyethylene glycol, based polyethylene glycol, using pegaso
Funders Acknowledgements:
National Institute of Arthritis and Musculoskeletal and Skin Diseases
Grant ID: UC2AR082196
Abstract
Innervation plays a critical role in osteoarthritis (OA) pain; however, its spatial organization and disease-related alterations remain poorly characterized, largely due to the technical challenges of imaging nerves in intact joints. This protocol outlines a step-by-step workflow for visualizing neurofilament-positive (NF⁺) nerve fibers in whole and bisected rat knees using a modified version of the organic solvent-based polyethylene glycol-associated solvent system (PEGASOS) tissue clearing method (Jing et al. 2018). This approach renders rat knee joints of varying thickness optically transparent, enabling high-resolution mapping of joint innervation.
Materials
Reagents:
| A | B | C | D | E | |
| Name | Vendor | CAS number | Catalog Number or Product Code | RRID | |
| Phosphate buffered solution (10X) | Thermo Fisher | BP39920 | |||
| Paraformaldehyde | Millipore Sigma | 30525-89-4 | P6148 | ||
| Sodium hydroxide (NaOH) | Millipore Sigma | 1310-73-2 | S5881 | ||
| Hydrochloric acid (HCL) | Fisher Scientific | 7647-01-0 | A144-500 | ||
| Immunocal Decalcifier | StatLab | 1214 | |||
| Triton X-100 | Millipore Sigma | 9036-19-5 | X100 | ||
| Bovine Serum Albumin (5% TBST) | Boston Bioproducts | IBB-169 | |||
| Normal Goat Serum | Abcam | ab7481 | |||
| Quadrol | Millipore Sigma | 102-60-3 | 122262 | ||
| Ammonium solution | Millipore Sigma | 1336-21-6 | 1.05432 | ||
| Tert-butanol | Millipore Sigma | 75-65-0 | 471712 | ||
| PEG-MMA500 | Millipore Sigma | 26915-72-0 | 447943 | ||
| Benzyl Benzoate | Millipore Sigma | 120-51-4 | B6630 | ||
| Anti-Neurofilament Antibody [NF421 + NFL/736) | Abcam | ab215903 | AB_3107022 | ||
| Goat anti-mouse Dylight 633 | Invitrogen | 35512 | AB_1307538 | ||
| Ammonium solution | Spectrum Chemical | 1336-21-6 | A-570 |
Troubleshooting
Collection and Fixation of Knee Joints
Perform transcardial perfusion using freshly prepared, ice-cold phosphate-buffered solution (PBS), followed by 4% paraformaldehyde (PFA) solution. Use approximately 250–300 mL each of PBS and 4% PFA for a 600 g rat. Indicators of successful perfusion include clear perfusate exiting the right atrium, blanching of the liver, and muscle tremors observed during the initial PFA flush.
Dislocate the hindlimb at the hip joint, carefully remove the muscles from the femur and tibia without disturbing the knee joint, excise the fibula, and trim the femur and tibia to the desired length.
Figure 1. Intact rat knee joint with muscles and fibula removed and bones trimmed approximately at mid-length.
Place the knee joint, with the tibia and femur aligned orthogonally as in Figure 1, in a labeled cassette and post-fix in 4% PFA at 4 °C for 24 hours.
Wash the knee joint with 1X PBS to remove residual fixative. Repeat this wash twice, allowing at least a couple of hours between washes or performing them over a few days. (Note: Collect the waste in a chemical hazard container and dispose of it following Institutional Environmental Health & Safety guidelines.)
Decalcification
Place the knee joints in a 50 mL tube containing approximately 40 mL of Immunocal solution (12–15 times the volume of the sample) and incubate at room temperature in a shaking incubator set to 100 RPM. Replace the Immunocal solution every 2–3 days. (Note: Complete decalcification of adult rat knees typically requires 7–9 days. Completeness of decalcification can be checked using CT features on an IVIS or with a Faxitron.)
After decalcification, replace the Immunocal solution with distilled water and place the samples in a shaker for at least two hours. Repeat this wash once to ensure thorough removal of residual decalcifying agent.
For half-joint preparations, bisect the decalcified knee in the sagittal plane using a sharp razor blade. For whole-joint samples, proceed directly to Step 8.
Figure 2. Decalcified rat knee joint bisected in sagittal plane.
Decolorization
Place the knee joint samples in Quadrol solution (25% w/v prepared in distilled water). Incubate at 37 °C in a shaker set to 100 RPM for 2 days, replacing the solution daily. Use approximately 20 mL of Quadrol per half knee and 40 mL for a whole knee joint.
Place the knee samples in 3% ammonium hydroxide solution and incubate at 37 °C in a shaker set to 100 RPM for 1 day
Immunolabeling
Rinse the sample(s) with 1X PBS.
Block the sample(s) overnight at 4 °C on a shaker using freshly prepared blocking buffer consisting of 1% Bovine Serum Albumin (BSA), 4% Normal Goat Serum (NGS), and 0.2% Triton X-100 in 1X PBS (PBST). Use approximately 3 mL of blocking buffer per half knee and 5 mL for a whole joint. (Note: Samples may be transferred to smaller containers such as 5 or 10 mL conical tubes or 20 mL scintillation vials. Ensure the sample is fully immersed.)
Incubate the sample(s) in primary antibody solution containing mouse anti-NF (1:500) diluted in staining buffer (4% Normal Goat Serum in 1X PBST) at 4 °C on a shaker for 7 days. Use approximately 3 mL of antibody solution per half knee and 5 mL for a whole joint.
Wash the sample(s) in PBST over one day. Begin with a quick rinse using 8–10 mL of PBST, followed by incubation in fresh PBST (8–10 mL) at 4 °C on a shaker for 3–4 hours. Repeat this wash cycle twice. (Note: Samples may be left in PBST overnight at 4 °C on a shaker if needed.)
Incubate the sample(s) in secondary antibody solution containing goat anti-mouse dylight 633 (1:400), diluted in staining buffer, at 4 °C on a shaker for 7 days. (Note: From this step onward, protect the sample(s) from light to prevent photobleaching.)
Wash the sample(s) in PBST over one day. Begin with a quick rinse using 8–10 mL of PBST, followed by incubation in fresh PBST (8–10 mL) at 4 °C on a shaker for 3–4 hours. Repeat this wash cycle twice. (Note: Samples may be left in PBST overnight at 4 °C on a shaker if needed.)
Delipidation and Clearing
Place the immunolabeled sample(s) in the gradient delipidation solution and incubate at 37 °C on a shaker set to 100 RPM. (Note: tert-butanol is solid at room temperature (melting point ~26 °C); melt it by placing the container in a beaker of warm water (~30 °C) before reconstituting it in distilled water.)
Incubate sample(s) in 30% tert-butanol with 3% Quadrol for 1 day. Use 15 mL of solution per half knee and 30 mL per whole knee.
Incubate sample(s) in 50% tert-butanol with 3% Quadrol for 1 day. Use 15 mL of solution per half knee and 30 mL per whole knee.
Incubate sample(s) in 70% tert-butanol with 3% Quadrol for 1 day. Use 15 mL of solution per half knee and 30 mL per whole knee.
Place the sample(s) in a solution of 70% tert-butanol, 30% PEGMMA500, and 3% Quadrol. Incubate at 37 °C on a shaker for 2 days, replacing the solution daily. Use 15 mL per half knee and 30 mL per whole knee. (Note: After this step, keep samples away from water-containing reagents.)
Place the sample(s) in BB-PEG medium (75% Benzyl Benzoate and 25% PEGMMA500) and incubate at 37 °C for 1 day. Use 15 mL of medium per half knee and 30 mL per whole knee.
Replace the BB-PEG medium after 1 day. Use 15 mL for half knee and 30 mL for whole knee.
(Note: Store sample(s) protected from light at room temperature until imaging.)
Figure 3. An entire rat knee joint post-clearing.
Figure 4. A bisected ( in sagittal plane) rat knee post-clearing.
Cleared sample(s) can be imaged using confocal and lightsheet microscopy techniques to visualize nerves.
Figure 5. An example of a PEGASOS-cleared rat knee (bisected in a sagittal plane) immunolabeled with neurofilament antibody (red). Green represents autofluorescence. The image was acquired using a MesoSPIM Lightsheet microscope.
Protocol references
1. Jing, D. et al. Tissue clearing of both hard and soft tissue organs with the PEGASOS method. Cell Res. 28, 803–818 (2018).
Acknowledgements
This work was funded by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (UC2AR082196). We thank the staff members at the Center for Biologic Imaging at the University of Pittsburgh for their assistance with 3D imaging.