From CLARITY Wiki
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| − | ==Non-perfused Tissue Samples== |
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CLARITY can still be performed on tissue samples that have not been perfused with hydrogel solution. |
CLARITY can still be performed on tissue samples that have not been perfused with hydrogel solution. |
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===Unfixed or Fixed Tissue Incubation=== |
===Unfixed or Fixed Tissue Incubation=== |
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The time period of incubation in the hydrogel monomer solution 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, like a whole mouse brain, 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 cells, causing less sites to be readily available for crosslinking with the formaldehyde and acrylamide hydrogel 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. |
The time period of incubation in the hydrogel monomer solution 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, like a whole mouse brain, 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 cells, causing less sites to be readily available for crosslinking with the formaldehyde and acrylamide hydrogel 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. |
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| − | ==Sample Storage== |
+ | ===Sample Storage=== |
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|Hydrogel Embedding]]). |
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|Hydrogel Embedding]]). |
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Revision as of 00:03, 5 April 2014
Tissue samples are placed into the hydrogel solution to incubate for several days in order for the hydrogel monomers to diffuse uniformly throughout the tissue before embedding via polymerization.
CLARITY can still be performed on tissue samples that have not been perfused with hydrogel solution.
Unfixed or Fixed Tissue Incubation
Whenever possible, it is best to replace the normal fixative solution with the hydrogel monomer solution, even when perfusion is not performed. However, tissue that has been stored in fixative solution, such as formalin, can also be processed using CLARITY. Place non-perfused tissue in the ice cold hydrogel solution (freshly-made or thawed) and incubate at 4°C in the refrigerator. Incubation can be on a shaker plate or standing still. The amount of hydrogel solution used should be enough to completely cover the tissue sample and provide a small excess to support monomer diffusion into the tissue.
Incubation Time
The time period of incubation in the hydrogel monomer solution 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, like a whole mouse brain, 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 cells, causing less sites to be readily available for crosslinking with the formaldehyde and acrylamide hydrogel 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 Storage
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).