Aug 06, 2024
Tooth Clearing with KneeEZ
This protocol is a draft, published without a DOI.
- Akash Gandhi1
- 1University of Michigan School of Dentistry
Protocol Citation: Akash Gandhi 2024. Tooth Clearing with KneeEZ. protocols.io https://protocols.io/view/tooth-clearing-with-kneeez-dikq4cvw
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: August 06, 2024
Last Modified: August 06, 2024
Protocol Integer ID: 104816
Keywords: clearing, EZ Clear, tooth clearing with kneeez kneeez, tooth clearing, optical transparency in the tooth, tooth sample, extracted tooth sample, calcified tooth structure, decalcification time, regards to decalcification time, tooth structure, tooth, kneeez kneeez, optical transparency, lectin labeling, blood vasculature morphology, neuronal ending
Funders Acknowledgements:
Joshua Emrick
Abstract
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.
Attachments
Materials
| Name | Vendor | Catalog Number | |
| 32% Paraformaldehyde Aqueous Solution, EM Grade | Electron Microscopy Sciences | 15714-S | |
| PBS, Phosphate Buffered Saline, 10X Solution, Fisher BioReagents | Fisher Scientific | BP3991 | |
| Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight 649 | Thermo Fisher | L32472 | |
| Invitrogen UltraPure 0.5 EDTA, pH 8.0 | Thermo Scientific | 15575020 | |
| Anti-beta III Tubulin antibody - Neuronal Marker | Abcam | #18207 | |
| Phosphate Buffer Solution | Sigma Aldrich | P3619 | |
| Glycine | Sigma Aldrich | G7126 | |
| Heptakis(2,6-di-O-methyl)-B-cyclodextrin | Santa Cruz Biotechnology | CAS 51166-71-3 | |
| Nycodenz - Iohexol | Progen | 18003 | |
| Tetrahydrofuran (THF), anhydrous, 250 ppm BHT added C3H8O FW=72.11 | Sigma | #186562 | |
| Urea (NH2CONH2)FW=60,06 | Sigma | #U5378 | |
| ddH2O | MiliQ | ||
| WHEATON liquid scintillation vial with attached cap | Millipore-Sigma | DWK986546 | |
| Nalgene vacuum filtration system filter, 0.2 um pore size | Sigma-Aldrich | Z370606 | |
| Hu-Friedy #½ DE Hollenback Carver with Regular Handle | Net 32 | CVHL1/2 |
Troubleshooting
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
Retro-orbital Injection (Day 1)
Anesthetize the mouse via induction at 5%
Toe pinch to ensure that the mouse is unresponsive
Wash samples with PBS (3x60 minutes) with gentle agitation at room temperature
Transfer to 10% w/v EDTA with gentle agitation at room temperature
Change the solution every two days for a combined total of 6 days
Wash decalcified teeth in PBS (3x1 hour) at room temperature with gentle agitation
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
Allow the mouse to regain consciousness and wait 5 minutes before beginning perfusion
This allows for vascular diffusion of lectin
Transcardial Injection and Perfusion (Day 1)
100 uL of Lectin DyLight 647 into the left ventricle followed by perfusion
Sample Harvest
Extract molars from mouse
View video example
Collect the sample in ice cold PBS
Drop-fix teeth in 4C PFA overnight at 4C on the shaker (gently agitation)
Decalcification (Day 2 through Day 8)
Wash samples with PBS (3x60 minutes) with gentle agitation at room temperature
Transfer to 10% w/v EDTA with gentle agitation at room temperature
Change the solution every two days for a combined total of 6 days
Wash decalcified teeth in PBS (3x1 hour) at room temperature with gentle agitation
Delipidation and RI Matching (Day 8 through Day 12)
Place teeth in 50% THF (diluted from 100% with MiliQH2O) at room temperature overnight with gentle agitation (shaker)
Use glass scintillation vials to avoid plastic degradation and continue to use for future steps
Incubate sample in 70% THF for 12 hours
Incubate sample in 80% THF for 12 hours
Incubate sample in 100% THF for 2x12 hours
All at room temperature with gentle agitation
Wash samples with MiliQH2O (4x1 hour) at room temperature
After washing, remove as much water in the sample as possible. This is to avoid H2O interaction with the next step (RI Matching)
Place the teeth in the fume hood for 5 minutes to allow water to dissipate
Incubate in EZ Clear solution for 48 hours at room temperature with gentle agitation
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
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
Immunohistochemistry (Day 13 through Day 26)
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
Incubate in blocking solution (1X PBS + 0.2% Triton X-100 + 10% DMSO + 6% Donkey/Goat Serum) at room temperature for 2 days
Wash samples in PtwH (1X PBS + 0.2% Tween-20) (2x1 hour)
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
Rabbit anti Beta-Tubulin-III (TUJ1), 1:500
Wash samples with PtwH at room temperature and exchange wash solutions 4-5 times until the following day
Incubate with secondary antibody in incubation solution (PTwH + 3% Donkey/Goat Serum + 1% cyclodextrin) at room temperature for 4 days
Goat anti-rabbit 555 (1:1000)
Wash in PtwH (3x2 hours) at room temperature
Wash with 1X PBS (3x1 hour) at room temperature
Wash overnight with 1X PBS at room temperature
Incubate in 1X PBS with 0.05% sodium azide for 24 hours at room temperature
2nd RI Matching (Day 27 through Day 28)
Wash samples with MiliQH2O (4x1 hour) at room temperature (does this need to happen post IHC, it’s not expressly written in the protocol?)
After washing, remove as much water in the sample as possible. This is to avoid H2O interaction with the next step (RI matching)
Place the teeth in the fume hood for 5 minutes
Incubate in EZ clear solution for 48 hours at room temperature with gently agitation
Protocol references
A modified version of the KneeEZ clearing protocol developed by Nele Haelterman's group