From CLARITY Wiki
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.
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). Keep the sample on ice.
- 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.