TITLE: Tooth Clearing with KneeEZ
AUTHORS: Akash Gandhi

[DESCRIPTION]
KneeEZ is a robust method for clearing highly calcified tooth structures. In the Emrick Lab, it can be used to clear extracted tooth samples. In this application, the #D visualization of neuronal endings can be achieved via immunolabeling. Additionally, blood vasculature morphology can be elucidated through lectin labeling. This protocol has been optimized for optical transparency in the tooth in regards to decalcification time.

[BEFORE_START]
Solution Preparation:

500 mL of 10% weight/volume EDTA
Measure out 268.7 mL of 0.5M EDTA
Add appropriate amount of 1X PBS solution to get final volume of solution to 500mL
Store at room temperature

Working Phosphate Buffer Solution (0.02M)
Add 50 mL of 1M Phosphate Buffer (PB) stock solution to 950 mL of MiliQ ddH2O
Store at 4C

EZ View Solution:
Obtain a 250 mL glass beaker, stir bar, hot plate, and thermometer
Pour 35 mL of 0.02 M PB solution into the beaker and adjust the solution temperature to 37C
In order to achieve this temperature change the hotplate heat setting to around 1.2
Add the stir bar to the solution at this point. As the solution becomes more viscous, slowly increase the bar’s spinning velocity
Measure out 52.5g urea and slowly add it to the stirring solution (over the course of 2-3 minutes). Then add 31.25 mg of sodium azide. There will be an immediate temperature drop after adding both solutes. Allow the temperature to equilibrate back to 37C and wait for complete dissolution 
Weigh 100g of Nycodenz iohexol
Vacuum filter and place in container
Adjust to final volume of 125 mL with 0.02 PB after solution is clear and filtered. Good at room temperature for up to 2 months

[STEPS]
SECTION: Retro-orbital Injection (Day 1)
1. Anesthetize the mouse via induction at 5%
SECTION: Retro-orbital Injection (Day 1)
1.1. Toe pinch to ensure that the mouse is unresponsive
SECTION: Retro-orbital Injection (Day 1)
6. Take the mouse out of the induction chamber and immediately inject 100 uL of Lectin DyLight 647 into both the left and right retro-orbital sinus
SECTION: Retro-orbital Injection (Day 1)
7. Allow the mouse to regain consciousness and wait 5 minutes before beginning perfusion
SECTION: Retro-orbital Injection (Day 1)
7.1. This allows for vascular diffusion of lectin
SECTION: Transcardial Injection and Perfusion (Day 1)
8. 100 uL of Lectin DyLight 647 into the left ventricle followed by perfusion

SECTION: Sample Harvest
9. Extract molars from mouse
SECTION: Sample Harvest
9.1. View video example
SECTION: Sample Harvest
10. Collect the sample in ice cold PBS
SECTION: Sample Harvest
11. Drop-fix teeth in 4C PFA overnight at 4C on the shaker (gently agitation)
SECTION: Retro-orbital Injection (Day 1)
2. Wash samples with PBS (3x60 minutes) with gentle agitation at room temperature
SECTION: Retro-orbital Injection (Day 1)
3. Transfer to 10% w/v EDTA with gentle agitation at room temperature
SECTION: Retro-orbital Injection (Day 1)
4. Change the solution every two days for a combined total of 6 days
SECTION: Retro-orbital Injection (Day 1)
5. Wash decalcified teeth in PBS (3x1 hour) at room temperature with gentle agitation
SECTION: Decalcification (Day 2 through Day 8)
12. Wash samples with PBS (3x60 minutes) with gentle agitation at room temperature
SECTION: Decalcification (Day 2 through Day 8)
13. Transfer to 10% w/v EDTA with gentle agitation at room temperature
SECTION: Decalcification (Day 2 through Day 8)
13.1. Change the solution every two days for a combined total of 6 days
SECTION: Decalcification (Day 2 through Day 8)
14. Wash decalcified teeth in PBS (3x1 hour) at room temperature with gentle agitation
SECTION: Delipidation and RI Matching (Day 8 through Day 12)
15. Place teeth in 50% THF (diluted from 100% with MiliQH2O) at room temperature overnight with gentle agitation (shaker)
SECTION: Delipidation and RI Matching (Day 8 through Day 12)
15.1. Use glass scintillation vials to avoid plastic degradation and continue to use for future steps
SECTION: Delipidation and RI Matching (Day 8 through Day 12)
16. Incubate sample in 70% THF for 12 hours
SECTION: Delipidation and RI Matching (Day 8 through Day 12)
17. Incubate sample in 80% THF for 12 hours
SECTION: Delipidation and RI Matching (Day 8 through Day 12)
18. Incubate sample in 100% THF for 2x12 hours
SECTION: Delipidation and RI Matching (Day 8 through Day 12)
18.1. All at room temperature with gentle agitation
SECTION: Delipidation and RI Matching (Day 8 through Day 12)
19. Wash samples with MiliQH2O (4x1 hour) at room temperature
SECTION: Delipidation and RI Matching (Day 8 through Day 12)
19.1. After washing, remove as much water in the sample as possible. This is to avoid H2O interaction with the next step (RI Matching)
SECTION: Delipidation and RI Matching (Day 8 through Day 12)
19.2. Place the teeth in the fume hood for 5 minutes to allow water to dissipate
SECTION: Delipidation and RI Matching (Day 8 through Day 12)
20. Incubate in EZ Clear solution for 48 hours at room temperature with gentle agitation
SECTION: Delipidation and RI Matching (Day 8 through Day 12)
20.1. Note 1: EZ Clear solution should be made at least the a couple of days prior to this step to ensure that the EZ Clear solution does not crystalize
SECTION: Delipidation and RI Matching (Day 8 through Day 12)
20.2. Note 2: after this step, the teeth will become transparent and difficult to visualize. We can incorporate agarose embedding to avoid this issue in the future. For now, marking the cap on the scintillation vial with the amount of teeth in each bottle will ensure that you know the amount of teeth you will need to image
SECTION: Immunohistochemistry (Day 13 through Day 26)
21. Incubate samples in permeabilization solution (1X PBS + 0.2% Triton X-100 + 20% DMSO + 0.3 M glycine + 0.05% sodium azide) at room temperature for 2 days with gentle agitation
SECTION: Immunohistochemistry (Day 13 through Day 26)
22. Incubate in blocking solution (1X PBS + 0.2% Triton X-100 + 10% DMSO + 6% Donkey/Goat Serum) at room temperature for 2 days
SECTION: Immunohistochemistry (Day 13 through Day 26)
23. Wash samples in PtwH (1X PBS + 0.2% Tween-20) (2x1 hour)
SECTION: Immunohistochemistry (Day 13 through Day 26)
24. Incubate with primary antibody of choice in primary antibody incubation solution (PTwH + 5% DMSO + 3% Donkey/Goat Serum + 1% cyclodextrin) at room temperature for 4 days
SECTION: Immunohistochemistry (Day 13 through Day 26)
24.1. Rabbit anti Beta-Tubulin-III (TUJ1), 1:500
SECTION: Immunohistochemistry (Day 13 through Day 26)
25. Wash samples with PtwH at room temperature and exchange wash solutions 4-5 times until the following day
SECTION: Immunohistochemistry (Day 13 through Day 26)
26. Incubate with secondary antibody in incubation solution (PTwH + 3% Donkey/Goat Serum + 1% cyclodextrin) at room temperature for 4 days
SECTION: Immunohistochemistry (Day 13 through Day 26)
26.1. Goat anti-rabbit 555 (1:1000)
SECTION: Immunohistochemistry (Day 13 through Day 26)
27. Wash in PtwH (3x2 hours) at room temperature
SECTION: Immunohistochemistry (Day 13 through Day 26)
28. Wash with 1X PBS (3x1 hour) at room temperature
SECTION: Immunohistochemistry (Day 13 through Day 26)
29. Wash overnight with 1X PBS at room temperature
SECTION: Immunohistochemistry (Day 13 through Day 26)
30. Incubate in 1X PBS with 0.05% sodium azide for 24 hours at room temperature
SECTION: 2nd RI Matching (Day 27 through Day 28)
31. Wash samples with MiliQH2O (4x1 hour) at room temperature (does this need to happen post IHC, it’s not expressly written in the protocol?)
SECTION: 2nd RI Matching (Day 27 through Day 28)
32. After washing, remove as much water in the sample as possible. This is to avoid H2O interaction with the next step (RI matching)
SECTION: 2nd RI Matching (Day 27 through Day 28)
33. Place the teeth in the fume hood for 5 minutes
SECTION: 2nd RI Matching (Day 27 through Day 28)
34. Incubate in EZ clear solution for 48 hours at room temperature with gently agitation