Structure-staining relationships in histochemistry and biological staining. Part 3. Some comments on the intentional and artifactual staining of lipids.

The choice, basis and calculation of certain numerical coefficients for describing chemical structures of such staining reagents as dyes, enzyme substrates and visualising agents as described. The general value of electric charge, conjugated bond number, Hansch pi values, and molecular or ionic weights is emphasised. Hansch pi values as indicators of hydrophobic character, are then used to analyse reagents giving rise to the staining of neutral lipids. Dyestuffs used to stain lipids all have high Hansch pi values (greater than or equal to +1.0), with superior stains, e.g. Sudan Black B and Oil Red O, having values of greater than or equal to +7.0. In keeping with this, conversion of the basic dyes Nile Blue and Brilliant Cresyl Blue, with Hansch pi values of -2.4 and -3.6, into their oxazone derivatives, with Hansch pi values of +4.4, and 3.6, generates lipid staining compounds. Also in keeping with this correlation of lipophilia with Hansch pi values greater than +1.0 is the occurrence of artifactual lipid staining in enzyme histochemistry. Such artifacts can arise for instance when using certain naphthyl substrates which give rise to naphthoic intermediate reaction products, or when tetrazolium salts as used as visualising agents, yielding formazans as final reaction products. The Hansch pi values of the naphthols and formazans generated histochemically typically fall into the lipophilic range. Another artifact of lipid staining is the staining of basophilic entities, such as cell nuclei, by fat stains which carry amino substituents. Calculation of the Hansch pi values for protonated (and hence cationic) derivatives of such dyes yields values typical of basic dyes.