1. Tissue is excised and immersed in fixative at 4°C as quickly as possible. If tissues have a minimum dimension > 5 mm, then slices of this width are prepared. This ensures that over the fixation period of 4-24 h (time dependent on the size of the tissue and on the antigen) all regions of the tissue are adequately fixed, and the antigen is retained uniformly.
2. Following fixation with paraformaldehyde, the tissue is washed in PBS prior to dehydration with IMS using a series of 50, 70, and 90%, and three absolute IMS changes (30 min each).
3. If the tissue is fixed with acid-ethanol, it can be immediately transferred to absolute alcohol.
4. From the final alcohol change, tissues are transferred to a mixture of polyester wax and alcohol (50:50) at 40°C and subsequently to a 75:25 mixture before changing to 100% wax. Infiltration times are dependent on the size of the tissue, but for the size recommended above, 2 h in each of the mixtures and 4 h (2 changes) in wax should suffice. For larger pieces of tissue, the infiltration times should be extended accordingly. However, it should be remembered that the longer the tissue remains at 40°C, the greater is the risk of antigen loss. Extended infiltration (>24 h) also encourages hardening of tissue, which makes sectioning more difficult.
5. Following infiltration, tissue is blocked out and allowed to set at room temperature.
6. Blocks are best stored at 4°C.
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