If two histological sections, of thickness nµ and 2nµ, and having the same apparent intensity of staining, are at equilibrium with dyebaths of concentration B1 and B2 respectively, the affinity of the histological substrate for the dye is, under denned conditions, given by the expression ΔF° = - RT In I/B, where B is either (i) B2 if the thinner section is stained to saturation, or (ii) B1 if B1 = 3B2. The affinity tends to be greater when measured in a weaker dyebath, as implied in method (ii).
Evidence is presented suggesting that the uptake of the basic dye Azure A by pancreatic basal chromidial substance, goblet cell mucin and intestinal epithelial cytoplasm follows a Langmuir adsorption isotherm to a first approximation, in that, at low dyebath concentrations, the uptake of dye is proportional to the concentration of dyebath with which the substrate is in equilibrium, while in strong dyebaths the uptake approaches a plateau. A deviation from the ideal Langmuir isotherm in dyebaths of moderate concentration, observed in epithelial cytoplasm and to a lesser extent in pancreatic basal chromidial substance, may have been due to interaction between neighbouring dye-binding sites, or to the presence in a single area of sites with different affinities for the dye.