Fixation

Fixation is not usually considered to be involved in explanations as to how dyes stain tissues. In reality, of course, fixation is intimately involved in staining. The reactions that occur during any staining process must involve tissue constituents in some fashion. So their preservation and treatment prior to staining must be factors to consider. Fixation, dehydration, clearing, and to a lesser degree, infiltration, may have an impact on the final product. However, the greatest impact from preparatory treatments undoubtedly comes from the chemicals used to denature the proteins during fixation.

It is as well to stress that the fundamental means of tissue preservation is to alter protein in some fashion. Unfixed tissues are quite avid for dyestuffs, as shown by the reduced staining times required for frozen sections with H&E. The overall effect of most fixative chemicals is to reduce this avidity to a degree. Some reduce it very little, some a lot.

We usually do not view it from this perspective. Although we know that formalin fixation reduces dye uptake, we think of fixation in neutral buffered formalin as the standard. To a large degree, in common practice, it has become the base line for reference. For that reason, we often rate other fixative chemicals in relation to the staining and preservation characteristics obtained with formalin. There is nothing wrong, or undesirable in this particularly. One base line for reference is just about as good as any other. We should keep in mind, however, that fixation does not usually increase dye uptake, rather it tends to diminish it.

Staining is so intimately associated with the preparatory techniques used that it would be foolish to discuss one without commenting on the other. While understanding the underlying principles involved in staining can be used to manipulate the process effectively, part of what must be understood is the effects of fixatives which may be used. These can have a very distinct impact on how a dye may stain a particular tissue.

There are numerous examples that can be given. No one who wanted to have good H & E staining of routine sections would use osmium fixation. Quite apart from the cost, the eosin staining would be terrible. On the other hand, it would be most appropriate to use an acetic acid containing fixative if the primary target for demonstration was the cell nucleus. With cytoplasmic granules as the primary target, it should probably be avoided.

It is the histotechnologist's responsibility to be aware of the most appropriate fixative to use, to know what staining techniques are most effective following fixation in particular mixtures, and to be aware of the likely results of unusual fixative/staining technique combinations.

 


 

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