TITLE: KneEZ Clear: an Effective Tissue Clearing Method for Intact Mouse Joints, including the Knee
AUTHORS: Julia Younis, Taeyong Ahn, Luis Tovias, Azeez Ishola, Brendan Lee, Joshua Wythe, Nele Haelterman

[DESCRIPTION]
KneEZ Clear is a modified version of the EZ Clear method (Hsu et al., 2022), optimized for clearing mouse musculoskeletal tissues. KneEZ Clear works well for the clearing of mouse knee and ankle joints, spines, temporomandibular joints, and teeth. 

Critical factors that impact the tissue’s optical transparency include fixation and decalcification time and temperature, as well as gradual lipid removal.

This protocol includes injection with fluorescently conjugated lectin to label the vasculature, followed by collection of tissue samples, fixation, tissue delipidation, and refractive index matching (e.g. tissue clearing). While lectin-labeled vasculature serves as a good positive control, it can be omitted when one only wants to look at endogenous fluorescence. In this case, proceed directly to transcardial perfusion of PBS and fixative. 

To efficiently label the microvasculature and venous endothelium, we employ a combinatorial approach of retroorbital perfusion followed by transcardial perfusion pre-fixation, followed by an additional pulse of diluted lectin post-fixation. This combination results in superior fluorescent labeling of the venous and capillary vasculature, as well as larger diameter arterial and venous vessels.



[STEPS]
SECTION: Retro-orbital Injection
1. Deeply anesthetize the mouse via inhalation of 4-5% isoflurane to induce anesthesia. Toe pinch to confirm animal is not receptive to painful stimuli. 
SECTION: Retro-orbital Injection
2. Using an insulin syringe, inject 50 µL of conjugated lectin to the retro-orbital sinus of the right and left eye. Allow the mouse to regain consciousness and mobility, and wait a minimum of 15 minutes before starting the transcardial perfusion procedure to allow lectin to move through the venous and capillary circulation.
 

SECTION: Transcardial Injection and Perfusion
4. On a Styrofoam board, fix the mouse in place by inserting 30-gauge syringes or needles into the paws and board.  
SECTION: Sample Harvest
17. Remove the skin from the lower half of the mouse. Be careful to not let hairs get on the sample. 
SECTION: Sample Harvest
18. Place mouse on its abdomen and have its dorsal side face you. With scissors or a razor blade, cut through the femur, close to the hip joint, to remove the hindlimbs from the rest of the body.

 

SECTION: Sample Harvest
19. Collect samples in ice-cold 1x PBS.
 

SECTION: Sample Harvest
20. Drop-fix the hindlimb samples in 4% PFA at 4 °C overnight (960 min) with gentle agitation

 


 

SECTION: Decalcification
21. Wash fixed samples with 1x PBS (3 times for60 min per wash) under gentle agitation.

SECTION: Decalcification
22. Transfer samples to 10% EDTA in PBS, and incubate at room temperature, under gentle agitation, for 2 to 5 days, changing the EDTA solution every other day.
 


SECTION: Decalcification
23. Wash decalcified samples with 1x PBS (3 times for 60 min) at room temperature under gentle agitation

SECTION: Lipid Removal
27. Place samples in 50% THF (in ddH2O) with triethylamine, incubate 1440 minat room temperature with gentle agitation.

50% THF + triethylamine (20 mL): 
10 mL THF, 20 uL triethylamine, adjust volume to 20 mL with ddH2O

 

SECTION: Lipid Removal
28. Sequentially incubate samples in 70% THF (x1), 80% THF (x1), and 90% THF (x2), for 720 min each step at room temperature with gentle agitation.

70% THF + triethylamine (20 mL):
14 mL THF, 30 uL triethylamine, adjust volume to 20 mL with ddH2O

80% THF + triethylamine (20 mL): 
16 mL THF, 44.8 uL triethylamine, adjust volume to 20 mL with ddH2O

90% THF + triethylamine (20 mL): 
18 mL THF, 64 uL triethylamine, adjust to 20 mL with ddH2O

 


SECTION: Lipid Removal
29. Remove THF and wash samples with ddH2O (4 times for 60 min) at room temperature with gentle agitation.

SECTION: Clearing
31. Incubate the tissue in EZ View solution at room temperature with gentle agitation for 
24-48 hours to render the tissue transparent. 
Important: Avoid cold temperatures to prevent the urea from crystallizing
SECTION: Imaging
32. If imaging using a Zeiss Z.1 LSFM system, trim excess muscle from the sample and mount in
1% filtered agarose made in ddH2O with a sample holder embedded at the top of sample after clearing. For this, you can use a 3 or 5 mL syringe with the top cut off for mounting as shown below. 

 

SECTION: Imaging
33. Once agarose solidifies, re-equilibrate in EZ View for an additional 1440 min at room temperature with gentle agitation. If using the Zeiss Z1 system, a minimum of 25 mL of EZ View is required for imaging. 
 


SECTION: Imaging
34. Store sample in EZ View solution before, during, and after imaging. 
SECTION: Expected Results
35. 
Below are two images of KneEZ Cleared adult mouse knee joints (12-14 weeks old):



 






SECTION: Clearing
30. Remove as much H2O as possible from the sample. 
 

SECTION: Transcardial Injection and Perfusion
5. Spray or wipe the ventral abdominal area of the mouse with 70% ethanol to sterilize the field and prevent fur from entering the chest cavity.
SECTION: Transcardial Injection and Perfusion
3. Deeply anesthetize the mouse via inhalation of 4-5% isoflurane to induce anesthesia. Toe pinch to confirm animal is not receptive to painful stimuli.
SECTION: Transcardial Injection and Perfusion
6. Use forceps to lift the skin, then make a lateral incision across the skin of the abdomen using scissors.
SECTION: Transcardial Injection and Perfusion
10. With small scissors, cut the diaphragm (in a crescent shape, along the ribs, careful to not accidentally puncture the lungs or heart) to expose the thoracic cavity.
SECTION: Transcardial Injection and Perfusion
9. Make a lateral incision across the parietal peritoneum to expose the liver and diaphragm.
SECTION: Transcardial Injection and Perfusion
7. Cut the skin down the midline of the mouse to the top of the thoracic cavity.
SECTION: Transcardial Injection and Perfusion
13. Allow the lectin to circulate for 1-2 minutes.
With small scissors, snip open the right atrium to allow for exsanguination in subsequent steps.
SECTION: Transcardial Injection and Perfusion
12. Using an insulin syringe, slowly pierce the left ventricular wall of the heart from the apex of the heart, being careful not to pierce the septum or enter the right ventricle and inject 100 µL
of conjugated lectin to the left ventricle. 
 

 

SECTION: Transcardial Injection and Perfusion
11. Make two upward cuts on the left and right side of the rib cage to create a flap that can be lifted to better access the heart. Using a hemostat, grip and secure the flap opening to provide access to the chest cavity.
SECTION: Transcardial Injection and Perfusion
15. Using a syringe pump set to a rate of 4 mL/min connected to a 25-gauge 3/4" winged infusion needle, inject 5 mL of room temperature, freshly prepared 4% PFA to the left ventricle to fix all tissues. Insert the needle into the puncture hold made in previous steps. Creating a new puncture may cause fluids to leak out.
 

SECTION: Transcardial Injection and Perfusion
16. Using a 27.5-guage needle, slowly inject 1 mL of diluted lectin (1:10 dilution in 1x PBS) to the left ventricle. Insert the needle in the puncture hole made in previous steps. 
SECTION: Methanol Pretreatment and Heme Removal
24. Dehydrate samples with 50% methanol in 1x PBS (x1), 80% methanol (x1), and 100% methanol (x2) for 60 min each step at room temperature with gentle agitation. 
SECTION: Methanol Pretreatment and Heme Removal
26. Sequentially rehydrate samples with 100% methanol (x2), 80% methanol in 1x PBS (x1), 50% methanol (x1), 1x PBS (x2) for 60 min each step with gentle agitation.
SECTION: Methanol Pretreatment and Heme Removal
25. Bleach samples with 5% H2O2 + 20% DMSO in methanol (1 vol 30% H2O2 / 1 vol DMSO / 4 vol methanol, ice cold) at4 °C for 960 min with gentle agitation to remove heme and decolorize tissue.
 

SECTION: Transcardial Injection and Perfusion
8. With forceps, pull back both flaps of skin to expose the peritoneum. 
SECTION: Transcardial Injection and Perfusion
14. Using a syringe pump set to a rate of 4 mL/min connected to a 25-gauge 3/4" winged infusion needle, inject 5 mL of warm (37°C) 1x PBS to the left ventricle to remove the blood. Insert the needle in the puncture made by the insulin syringe (if possible) while stabilizing the heart with blunt forceps. Make sure the needle is angled towards the aorta to prevent lung inflation. When the fluid exiting the mouse is clear of blood, proceed to the next step.