Dec 06, 2019
Tissue Preparation for CLARITY
- 1University of Florida
- Optical Clearing of Tissue
- Human BioMolecular Atlas Program (HuBMAP) Method Development Community
External link: http://wiki.claritytechniques.org/index.php/Solutions
Protocol Citation: Seth Currlin, Marda Jorgensen, Jerelyn Nick 2019. Tissue Preparation for CLARITY. protocols.io https://dx.doi.org/10.17504/protocols.io.8jihuke
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: October 22, 2019
Last Modified: December 06, 2019
Protocol Integer ID: 29002
Keywords: CLARITY, Hydrogel Solution, Clearing Solution, tissue fixation, paraformaldehyde, PFA, acrylamide, lipid clearing
Abstract
Tissue preparation for CLARITY includes fixation in 4% PFA, infusion with monomers in Hydrogel Solution, and clearing of lipids in Electrophoretic Tissue Clearing Solution.
Guidelines
The hydrogel solution components should be kept on ice during preparation to prevent polymerization. The thermal initiator is stable at low temperatures, but initiates polymerization at higher temperatures. To prevent polymerization, the hydrogel solution aliquots should be stored at -20°C until ready for use. However, the solution should be stable at 4°C for a few days and at room temperature for a couple hours.
Materials
Hydrogel Solution
| Ingredient | Amount | Final Concentration | Purpose | |
| 40% Acrylamide | 40 mL | 4% | Hydrogel network monomer | |
| 2% Bis-acrylamide | 10 mL | 0.05% | Small chemical crosslinker | |
| VA-044 Initiator | 1 g | 0.25% | Polymerization thermal initiator | |
| 16% Paraformaldehyde | 100 mL | 4% | Biomacromolecule crosslinker | |
| 10X PBS | 40 mL | 1X | Salt buffer | |
| Deionized water | 210 mL | - | Aqueous solvent |
Hydrogel Solution
Clearing Solution
Electrophoretic Tissue Clearing Solution C13001; logosbio.com,
Equipment:
Platform rocker at room temperature (PFA Incubation)
Rocking Stage at 4˚C (hydrogel solution incubation)
Incubator Shaker/ at 37˚C (hydrogel polymerization)
Incubator/Shaker at 45˚C (tissue clearing)
Safety warnings
Paraformaldehyde and acrylamide are both toxic; therefore, preparation and use of the hydrogel solution should be done in a fume hood.
1. PFA Incubation: Trimmed tissues are placed fixed in 4% PFA for 20-24 hours at room temperature on a slow platform rocker.
2. Hydrogel Incubation: Tissues are transferred to Hydrogel Solution in for approximately 5 days at 4˚C. Tubes are kept on a 2-axis rocker for the duration.
3. Hydrogel Polymerization: On day 5 the tissue is placed in pre-warmed 37˚C PBS and a layer of mineral oil to deter oxygen exchange from air within tube. The hydrogel within the tissue is polymerized at 37°C for 4 hours with gentle rocking.
4. Passive Clearing: Following polymerization the tissue is placed in Clearing Solution and kept at 45˚C with gentle rocking until optically clear.