Perfusion
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Transcardial perfusion of the animal using the hydrogel solution in place of normal fixative is 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. Perfusiion 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. |
Transcardial perfusion of the animal using the hydrogel solution in place of normal fixative is 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. Perfusiion 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. |
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===Perfusion Procedure=== |
===Perfusion Procedure=== |
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| − | The following steps are used for transcardial perfusion of a mouse. The steps can easily be adapted to alternative protocols (i.e. for a rat) by replacing PFA with hydrogel solution. |
+ | 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. |
#Make or thaw the hydrogel monomer solution. Gently invert to mix, but avoid introducing bubbles. Keep the solution on ice until ready for use. |
#Make or thaw the hydrogel monomer solution. Gently invert to mix, but avoid introducing bubbles. Keep the solution on ice until ready for use. |
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#Prepare perfusion materials in a fume hood, and deeply anesthetize the mouse. |
#Prepare perfusion materials in a fume hood, and deeply anesthetize the mouse. |
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#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. |
#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. |
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| − | #Immediately |
+ | #Extract the tissue (e.g. brain) and place it immediately into the remaining ice cold hydrogel solution (~20 mL). |
| + | #Incubate the sample at 4 |
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Revision as of 05:24, 27 November 2013
Transcardial Perfusion
Transcardial perfusion of the animal using the hydrogel solution in place of normal fixative is 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. Perfusiion 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.
- Make or thaw the hydrogel monomer solution. Gently invert to mix, but avoid introducing bubbles. Keep the solution on ice until ready for use.
- Prepare perfusion materials in a fume hood, and deeply anesthetize the mouse.
- 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.
- Extract the tissue (e.g. brain) and place it immediately into the remaining ice cold hydrogel solution (~20 mL).
- Incubate the sample at 4