Solutions

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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.

Contents

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.
  • Changing acrylamide/bis-acrylamide 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. In contrast, decreasing acrylamide or bis-acrylamide concentration will loosen the network, causing it to be more porous but also weaker. Significant decreases in acrylamide concentration (0-1% acrylamide) will lead to more substantial protein loss during clearing.

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