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CLARITY Solutions

Two chemical solutions need to be prepared for CLARITY execution. The hydrogel solution contains the monomers necessary to form a hydrogel within the tissue sample. The clearing solution is used to remove the unattached lipids from the tissue sample following hydrogel embedding. In addition to the two prepared solutions, two other solutions are used to prepare the cleared tissue samples for imaging. PBST (0.1% TritonX in 1X PBS) is used to wash out the clearing solution micelles prior to either immunostaining or imaging. A mounting solution is used to match the internal refractive index of the cleared tissue in order to allow optimal transparency and penetration depth of the microscope during imaging. The different solutions used to incubate the tissue sample throughout CLARITY processing are described in the table below.

Solution Purpose Safe for Sample Storage? Sample Appearance
Hydrogel solution Infuses the tissue with monomers that covalently link to biomolecules and form a hydrogel polymer network No, but safe for sample incubation at 4°C for up to 2-3 weeks Opaque/No change
Clearing solution Removes light-scattering lipids from crosslinked tissue using micelles Yes, store at room temperature (or higher to speed up passive clearing) Swells as the sample clears, appears see-through, but may remain cloudy
PBST Intermediate buffer between clearing solution and mounting solution, also used for staining Yes, store at room temperature or 4°C Remains swollen, appears more cloudy than in clearing or mounting solution
Mounting solution Matches the cleared tissue refractive index to maximize transparency for imaging Not recommended Visually transparent, anatomical size

Tissue samples are swollen in the different solutions during CLARITY processing in the following manner:

Hydrogel solution ⇒ Clearing solution ⇔ PBST ⇔ Mounting solution

After imaging, the cleared tissue sample can be returned to PBST for storage or as an intermediate before being placed back in clearing solution for additional clearing or to wash out the first round of staining.

FocusClear is the best mounting solution for larger samples, such as the whole mouse brain. Alternative mounting solutions are discussed in the section below.

Hydrogel Solution

Components and preparation

The following table summarizes the ingredients of the hydrogel solution and their purpose. Amounts are listed for a 400 mL batch of 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

The hydrogel solution components should be kept cold on ice during solution preparation to prevent polymerization. Paraformaldehyde and acrylamide are both toxic chemicals, so all preparation and handling of the hydrogel solution should be performed with personal protective equipment in a fume hood. Following preparation of the stock solution, 40 mL aliquots can be distributed into 50 mL conical tubes, also kept on ice. This volume is ideal for the hydrogel embedding of a mouse brain sample, which requires 20 mL of hydrogel solution for perfusion and 20 mL for incubation and embedding (one tube per mouse brain). Larger aliquots may be needed for alternative animals or tissues, such as the perfusion of a rat. A good rule of thumb is to prepare the same volume of hydrogel solution as would normally be used for fixative solution. The hydrogel solution is essentially replacing the pure fixative, such as 4% PFA, with an enhanced fixative containing both PFA and the components necessary for hydrogel embedding.

Storing the hydrogel solution

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.

Changes to the hydrogel solution composition

The standard hydrogel solution has been optimized to provide a balance between hydrogel rigidity and porosity with minimal protein loss after clearing. However, solution adjustments may be useful for applying CLARITY to certain kinds of tissues.

  • Removing bis-acrylamide - Bis-acrylamide is a small molecule that directly crosslinks polyacrylamide chains to form a gel. It is a secondary crosslinker to the biomolecules inside the tissue that chemically link to polyacrylamide via formaldehyde, thus acting as the primary crosslinker. Bis-acrylamide increases the rigidity of the hydrogel network by creating crosslinks inside spaces that may be void or sparse of biomolecules. The presence of bis-acrylamide also causes all the hydrogel solution surrounding the tissue sample to crosslink and form a gel during the embedding step, which then has to be manually removed from the tissue via physical rubbing/handling. Removing bis-acrylamide (replacing with water) prevents gelation from occurring outside of the tissue, so the sample can easily be removed from the solution following the embedding step. This is recommended for small or fragile samples that cannot withstand the physical gel removal process.
  • Increasing monomer concentration - Increasing the concentration of either acrylamide or bis-acrylamide in the hydrogel solution will result in a stiffer, stronger hydrogel. The trade-off is a loss in porosity (limited diffusion) due to the tighter polymer network that is formed. As a result, the tissue will likely need longer time periods for clearing.
  • Decreasing monomer concentration - Decreasing acrylamide or bis-acrylamide concentration will loosen the network, causing it to be more porous but also weaker. Decreases in acrylamide concentration (0.5-1% acrylamide) will lead to more substantial protein loss during clearing because fewer biomolecules will be chemically attached to the hydrogel network. Still, preliminary studies show that enough biomolecules are maintained to get good signal from labelled antibodies present before clearing as well as those introduced via immunostaining after clearing. The gain in porosity of samples embedded in these lower concentrations of acrylamide significantly decreases the duration of diffusion-limited steps such as passive clearing and immunostaining. Note that lower acrylamide concentrations may not gel outside of the tissue during the polymerization step as the bis-acrylamide concentration may be too low to form a crosslinked network. A hydrogel matrix will still form inside the tissue due to the added crosslinking from the biomolecules.

Clearing Solution

Components and preparation

The following table summarizes the ingredients of the clearing solution and their purpose. Amounts are listed for a 10 L batch of solution.

Ingredient Amount Final Concentration Purpose
Sodium dodecyl sulfate 400 g 4% Lipid clearing surfactant
Boric acid 123.66 g 200 mM pH buffer
Sodium hydroxide To pH 8.5 - pH adjustment ions
Deionized water Fill to 10 L - Aqueous solvent

A large amount of clearing solution is used during electrophoretic tissue clearing (4-5 L), so having a large volume prepared is not unreasonable. The easiest way to make this solution is to first prepare a solution of boric acid in 2 L of water and add NaOH to adjust the pH to 8.5. Then, add the boric acid solution to a container with the SDS and fill to 10 L with water. Either SDS powder or concentrated solution, such as 20% SDS, can be used, but make sure to prepare the solution in a fume hood when using powder to avoid skin contact or inhalation. The clearing solution can be made and stored at room temperature.

Mounting Solution

Cleared tissue samples are placed in a mounting solution prior to imaging. This solution optimizes the sample transparency by matching the internal refractive index of the clarified tissue. Additionally, the mounting solution reverses the hydrogel swelling that occurs in the clearing solution and PBST, such that the sample returns to its anatomical size. For imaging, a sealed chamber containing the sample and the mounting solution is crafted in order to keep the sample swollen in its most visually transparent state.

Mounting solution options

Several different mounting solutions have been tested with varying results. The choice of mounting solution may effect how visually transparent the sample appears and how deep into the clarified tissue the microscope can image.

  • FocusClear - A commercially available solution with a refractive index of 1.45, FocusClear seems to provide the best transparency of clarified tissue samples. Fluorescence signal can still be measured even at deep penetration depths, which is ideal for thick samples such as a whole mouse brain. No damage to cellular structural features is apparent. If the sample has not been washed well in PBST, an irreversible white precipitate can develop within the embedded tissue after a few days, possibly from a reaction to leftover SDS.
  • Glycerol - 85% glycerol can be used as a mounting solution, however, it is not ideal for thicker samples. Some cloudiness may persist which will limit the achievable imaging depth. The glycerol concentration may need to be adjusted for different tissue types to get the best transparency.