Equipment and reagents
• Disposable moulds (Raymond A. Lamb)
• Tissue-Tek OCT compound (Bayer Diagnostics)
1 Before dissection, place a small amount of iso-pentane in a small Pyrex dish."
2 Place the dish in a polystyrene box containing a layer of 'dry ice' (C02).
• Freshly obtained mouse or human tissue
Protocol 5 continued
3 Cover box to lower temperature quickly.
4 Fill a mould" with Tissue-Tek, ensuring as few air bubbles as possible.
5 Place the tissue carefully in mould.c excluding air bubbles, and orientating it so that the required plane faces the bottom of the mould as this is the face that is presented to the ciyostat blade.
6 Add more Tissue-Tek, if required, to ensure the tissue is completely surrounded.*1
7 Place the mould in the iso-pentane and leave to freeze for at least 5 min.
a Do NOT use alcohol as it is miscible with Tissue-Tek and will not allow the block to freeze properly. It will then be impossible to cut as the texture will never harden to the same consistency as the tissue.
b Moulds can be plastic, metal, or foil. The foil mould is made by wrapping foil around an object, such as the end of a pen and sealing one end. Tap on the bench to flatten the end, then remove the pen.
c Upon dissection the tissue may be washed in PBS, but any fluid should be absorbed on a piece of tissue. Tissue-Tek will not adhere to wet material, which will detach from the block whilst cutting, therefore drying of the tissue is essential.
"This eliminates the possibility of'freezer-burn' if the blocks are to be stored for some time.
Protocols 6 and 7 outline one method for the perfusion of tissue in intact animals, prior to tissue dissection. The buffered lysine and paraformaldehyde used for perfusion can be prepared and stored at 4°C overnight, but they should only be mixed together when needed as the fixative does not keep (see Protocol 6). If the tubing becomes detached from the heart during the perfusion procedure it is not easy to reattach without losing fluid from the old incisions. Thus, fixation will be incomplete and the procedure should be abandoned. Care should also be taken so the line is not pushed through the heart and out the other side!
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