ABSTRACT

In immunocytochemistry, the fixation and tissue pretreatment must satisfy several mutually contradictory criteria: retention of antigens, preservation of antigenicity, preservation of structure, and allowing of permeability of tissue for antibodies and reporter molecules in pre-embedding studies. The essential features of the ultracryotomy technique involve fixation, cryoprotection, freezing, cutting, thawing, staining, and contrasting-drying. Even with insoluble antigens, however, fixation is usually necessary in order to allow structural preservation. With very few exceptions, most antigens are soluble in buffers and have to be chemically fixed or cross-linked in the tissue. Whenever possible, fixation should be undertaken by perfusion or by vapor-fixation of freeze-dried tissue blocks. Although generally a mild fixation, formalin or paraformaldehyde fixation can lead to destruction of certain epitopes. Finally, the type of treatment intended for the tissue after fixation is important, as mild fixatives such as weak paraformaldehyde solutions will not permit the tissue to go undamaged through harsh embedding conditions.