The Chemistry of Fixation and Staining: II. A Unified Theory of Acid/Base Staining Reactions

A new theory, based on the hard and soft acid and base (HSAB) principle, is proposed to explain interactions among two (or more) dyes and tissues. Ions lie at specific loci on a scale of hardness/softness (H/S), depending on the spatial nature of their charge site. Oppositely charged compounds of similar H/S characteristics form stable bonds which are difficult to break. Oppositely charged ions of dissimilar H/S characteristics form less stable bonds which are easily broken. A dye-tissue salt will be displaced if a second dye can form a better matched salt with either the first dye or the tissue.Through trial and error, iterative procedures, the authors have determined relative H/S values for 19 anionic dyes and 7 tissue components. Reactions among these dyes are completely predictive, given the H/S value of a tissue cation. This theory should lead to much greater understanding of general and many special staining procedures which utilize anionic and cationic dyes.