From CLARITY Wiki
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| − | The hydrogel solution components should be kept cold on ice during the solution preparation to prevent polymerization. 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). |
+ | The hydrogel solution components should be kept cold on ice during solution preparation to prevent polymerization. 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 you would normally use of 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=== |
===Storing the hydrogel solution=== |
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Revision as of 22:16, 4 October 2013
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. 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 you would normally use of 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.