From CLARITY Wiki
Tissue samples are placed into the hydrogel solution to incubate for several days. This allows the hydrogel monomers, formaldehyde and acrylamide, to diffuse uniformly throughout the tissue. Perfusion is recommended because it introduces the monomers inside the tissue prior to incubation.
Importance of Hydrogel Solution Incubation
Allowing ample time for the hydrogel monomers to distribute uniformly throughout the tissue sample is important for incorporating biomolecules into the hydrogel network. Once inside the tissue, formaldehyde reacts with the primary amine groups present on many biomolecules, including proteins, DNA, and RNA. Following this reaction, formaldehyde reacts with acrylamide, thereby covalently linking the tissue biomolecules to the acrylamide monomers that will be polymerized to form a hydrogel mesh within the tissue. Infusing formaldehyde and acrylamide into the tissue together promotes the biomolecule-acrylamide linking. Biomolecules attached to multiple acrylamide groups act as crosslinks for the hydrogel once acrylamide polymerization occurs.
It is important to note that only biomolecules with primary amine groups available will be incorporated into the hydrogel and therefore maintained after lipid clearing. Tissue elements with a greater number of amine groups are more likely to react with the monomers and be attached to the hydrogel network. It is possible that biomolecules with very few primary amine groups will undergo little to no incorporation and be washed out during clearing.
Changes to the hydrogel solution composition may affect biomolecule incorporation into the hydrogel. For instance, lowering the acrylamide or PFA concentration will lower the number of biomolecule-acrylamide attachment points, likely resulting in greater protein or DNA/RNA loss during lipid clearing. Such possible consequences should be kept in mind when altering the hydrogel solution composition, and they may or may not affect subsequent visualization of the maintained structural features.
Fixed tissue samples have already undergone crosslinking via formaldehyde, thus reducing the number of primary amine sites available to react with the hydrogel monomers. For this reason, increased hydrogel solution incubation times are used with these samples in order to allow additional time for formaldehyde and acrylamide to seek out unused amine sites for hydrogel linking. The results on fixed human samples following CLARITY processing, which show observable structural features after immunostaining, indicate that enough unused amine sites remain on the tissue biomolecules for sufficient incorporation into the hydrogel network.
- Perfused tissue samples - The transcardial perfusion procedure introduces hydrogel solution inside the tissue via the blood vessels, so less incubation time is needed for the monomers to uniformly distribute throughout the tissue. Following perfusion, the extracted tissue sample is placed in hydrogel solution (~20 mL for a whole mouse brain) for 2-3 days.
- Non-perfused tissue samples - The time period of hydrogel solution incubation for non-perfused tissue samples depends on the size of the sample and whether it was previously fixed. For unfixed tissue, smaller samples (e.g., less than 1-2 mm thick) may only need 2-3 days of incubation before embedding, while larger samples may require longer soaking time, such as 5-7 days or more. Fixed tissue samples have already undergone crosslinking of many of the primary amine sites within the tissue, causing less sites to be readily available for crosslinking with the formaldehyde and acrylamide monomers. Longer incubation times, such as 1-2 weeks, should be used for samples previously stored in fixative to allow extra penetration time for the monomers to diffuse into the crosslinked tissue and find the unused primary amine sites for hydrogel attachment.
Sample Incubation Conditions
Place tissue in the ice cold hydrogel solution (freshly-made or thawed) and incubate at 4°C in the refrigerator or cold room. Incubation can be on a gentle shaker plate or standing still. The amount of hydrogel solution used should be enough to completely cover the tissue sample and provide an excess to support monomer diffusion into the tissue.
- Note: It is always best to keep incubating samples near the back of the refrigerator to keep them as cold as possible. Samples placed near the door can start to polymerize after just a few days.
Tissue samples should not be stored in the hydrogel solution at 4°C for more than 2-3 weeks. The thermal initiator in the hydrogel solution is not stable for long periods of time at this temperature, and acrylamide polymerization can slowly occur. It is undesirable to let polymerization occur uncontrolled over time as it could lead to an excessive formation of crosslinked polyacrylamide inside the tissue, rendering it rigid and difficult to clear (see Hydrogel Embedding).