Perfusion

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#Incubate the sample at 4°C for 2-3 days to allow further diffusion of the hydrogel solution into the tissue.
 
#Incubate the sample at 4°C for 2-3 days to allow further diffusion of the hydrogel solution into the tissue.
 
*''Note:'' A large syringe or a perfusion pump can be used to deliver PBS and the hydrogel solution during the perfusion. If using a perfusion pump, be sure to rinse the tubing thoroughly with water afterwards to prevent polymerization inside the tubing over time, which could result in blockages.
 
*''Note:'' A large syringe or a perfusion pump can be used to deliver PBS and the hydrogel solution during the perfusion. If using a perfusion pump, be sure to rinse the tubing thoroughly with water afterwards to prevent polymerization inside the tubing over time, which could result in blockages.
 
 
==Non-perfused Tissue Samples==
 
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|Hydrogel Embedding]]).
 

Latest revision as of 19:34, 4 April 2014

Transcardial perfusion of the animal using the hydrogel solution in place of normal fixative is highly recommended, but not essential. By adding acrylamide monomers into the tissue at the same time as paraformaldehyde (PFA), it is easier for the acrylamide to attach to the biomolecules inside the tissue. Perfusion also quickly introduces hydrogel solution uniformly throughout the tissue so that passive diffusion of the monomers over time is not the only method of penetration.

[edit] Perfusion Procedure

The following steps are used for transcardial perfusion of a mouse. The steps can easily be adapted to alternative protocols (e.g. for a rat) by replacing PFA with hydrogel solution.

  1. Make or thaw the hydrogel monomer solution. Gently invert to mix, but avoid introducing bubbles. Keep the solution on ice until ready for use.
  2. Prepare perfusion materials in a fume hood, and deeply anesthetize the mouse.
  3. Perfuse the mouse transcardially with 20 mL of ice cold PBS followed by 20 mL of the ice cold hydrogel solution at a slow rate of about 10 mL/min.
  4. Extract the tissue (e.g. brain) and place it immediately into the remaining ice cold hydrogel solution (~20 mL). Keep the sample on ice.
  5. Incubate the sample at 4°C for 2-3 days to allow further diffusion of the hydrogel solution into the tissue.
  • Note: A large syringe or a perfusion pump can be used to deliver PBS and the hydrogel solution during the perfusion. If using a perfusion pump, be sure to rinse the tubing thoroughly with water afterwards to prevent polymerization inside the tubing over time, which could result in blockages.