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.

Hydrogel Solution

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.