Perfusion

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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.
 
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===
 
===Incubation Time===
The time period of incubation in the hyrogel 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.
+
The time period of incubation in the hyrogel 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 the primary amine sites within the cells.
 
==Sample Storage==
 
==Sample Storage==

Revision as of 01:27, 20 February 2014

Contents

Transcardial Perfusion

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.

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.

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 hyrogel 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 the primary amine sites within the cells.

Sample Storage