Tissue State

Often, when discussing staining, we focus on the dyes and other chemicals we use in a particular technique. Equally or more important is the reactivity of the tissue that we are staining. There are several factors which may affect the condition of these tissues in relation to the dyes used in staining.

We often do not consider the effect of fixation on staining, but it does have a significant impact. Some fixatives inhibit staining, while others help facilitate it. The choice of fixative can have a significant impact on both the depth of staining achieved and on subsequent manipulations of it.

In the specific context of trichrome stains fixatives containing mercuric chloride and/or picric acid are the ones of choice. Both fixing agents improve staining by acid dyes. Extended fixation in formal sublimate has been recommended for the more difficult methods, used for the demonstration of fibrin.

Reasonably satisfactory staining of material fixed in formalin variants can be obtained with some methods by altering the times of each step, but secondary fixation of sections in Bouin's fluid or saturated aqueous picric acid for an hour at 56°C is recommended for routine methods whenever possible.


Tissue accessibility
Obviously, before chemicals, including dyes, can react with tissue they must be able to get there. In some cases the tissue itself inhibits this. There are several reasons why this can be so.

A common cause is the use of an aqueous solvent and a hydrophobic tissue component. It has been suggested that some fibres are coated or intimately associated with lipids of some kind, and these lipid materials inhibit access by aqueous solutions. There are two steps where this can seriously impact on staining. The first is applications of dyes themselves, but perhaps equally important is the inhibition of aqueous fixatives and reduced fixation as a consequence. A process called degreasing has been developed to eliminate both of these problems for methods where fixation and dye application are critical. Other reasons might be the physical state of the tisse, such as dense bone or tendon, which physically inhibits any penetration by dyes. All of these inhibit access of the dye to the tissues, and require increased time to accomplish staining.

Of course, the converse can be true as well. Some tissues can be easily accessed. Collagen is a good example. It is easily stained, and requires little time to accomplish. Between the extremes are a wide variety of "textures", so to speak, and a correspondingly wide variety of staining results and intensities.


Tissue density
Tissue density refers to the number of dye binding sites a particular tissue has in a given volume. That is, how much dye will attach to it compared to the amount that will attach to other tissues? Since we are talking about the attachment of acid dyes to tissue, we are really talking about the number of charged amino groups available. Tissues vary in this.

Obviously, if there is more dye attached to a particular structure it will appear more deeply stained. It will also take longer for the dye there to be replaced, and that can be important. Should dye be removed equally from all tissues, those components that have more will retain some dye when other components have been completely decolourised.



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