4.1.1 Frozen versus perfused tissue
Tissue may either be taken fresh from the animal and frozen immediately, or the animal may be perfused with fixative and then dissected. Perfused tissue yields better morphology than fresh tissue, however, the procedure requires practice to ensure the best result. Also, one is limited to a single fixative, whereas with fresh tissue it is possible to use a number of different fixatives to ensure optimal conditions for antibody binding.
Tissue processed by wax embedding methods may also be used, but generally the stages of alcohol dehydration and the temperature required for infiltration and embedding render the tissue unsuitable for all but the most robust antibody staining.
4.1.2 Practical considerations for the preparation of fresh tissue
It is important to harvest and freeze tissue as quickly as possible to preserve antigen. If processing more than one animal, sacrifice individually and dissect immediately. It is also preferable to use some form of cryo-protectant around material to be frozen as this allows for a longer storage time without marked deterioration of the tissue. Cryo-protectants may vary in quality and storage ability, but the example given (see Protocol 5) has proved satisfactory over a long period of time.
Freezing the tissue in an iso-pentane bath is preferable, though liquid nitrogen may be used. However, with liquid nitrogen freezing generally occurs too quickly, which may result in the distortion and cracking of the material. In an iso-peniane bath, the block freezes from the bottom leaving a small area at the top to freeze last, which allows for the expansion or contraction of the material. Once frozen, the tissue may be left for some months, but will eventually lose moisture and change texture, which makes cutting more difficult. A procedure for preparation of fresh tissue for immunohistochemistry is given in Protocol 5.
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