Methods are proposed for the estimation of the rate, activation energy, heat, and affinity of staining of histological sections, and approximate results are given for the staining of mucin, mast-cell granules, chromatin, cytoplasmic ribonucleic acid (RNA), cartilage matrix, and other structures by azure A.
The half-staining time t½ is the time taken by a substrate under given staining conditions to achieve half the intensity of staining it would reach at equilibrium, and is approximately equal to the time taken to stain in a given, fairly dilute dyebath to the same intensity as at equilibrium in a dyebath of half the given concentration. The activation energy E of staining is given by E = ln t½(1)/t½(2) x RT1T2/(T2-T1), where t½(1) and t½(2) are the half-staining times at absolute temperatures T1 and T2 respectively, and R is the gas constant. The activation energy of staining reflects the effect of temperature on rate of staining, and may be regarded as an index of substrate permeability. Half-staining times and activation energies of staining with azure A increase in the order mucin, mast-cell granules, chromatin, RNA, and interstitial cartilage matrix. Times of half-destaining and activation energies of destaining also are probably largely determined by substrate permeability. Differential staining dependent on differences in rate of staining may be enhanced by the use of chilled and stirred dyebaths, and by the use of dyes of large particle size.
The heat of dyeing δH, sometimes regarded as the sum of the heats of formation of the various dye-substrate bonds, approximately equals RT1T2/(T2-T1)x ln [D]1/[D]2, where [D]1 and [D]2 are the concentrations of dyebath giving equal intensity of staining of the substrate at equilibrium at temperatures T1 and T2. Approximate figures for δH in kcal/mole for staining with dilute azure A are: mucin, -8; chromatin and cartilage matrix, -7; cytoplasmic RNA, -5.5; mast-cell granules, - 2 to - 4. The higher the value of -δH the more is staining inhibited by a rise in temperature of the dyebath.
The affinity of a dye for a substrate may be regarded as the standard free energy change accompanying the staining process, which under certain conditions is given approximately by δF° = - RT ln τ /(1-τ)[D], where τ is the fraction of available staining sites in the substrate occupied by dye when the substrate is at equilibrium with a dyebath of concentration [D]. Differential staining of substrates with a high affinity for the dye is facilitated by the use of dilute dye solutions. Approximate values of δF° for staining with azure A at 4° C and pH 4.0, in kcal/mole, are: cartilage matrix, -3.8 (orthochromasia) and - 5.3 (metachromasia); mast-cell granules, -4 (orthochromasia) and -4.4 (metachromasia); RNA, -3.1; mucin, between - 2.7 and -3.4; chromatin, -3.1; thyroid colloid, -2.3; Xenopus poison gland secretion, -2.3 It is suggested that part of the high affinity of sulphate groups for basic dyes is due to an increase in entropy during staining, resulting from dispersion of a large hydration shell surrounding the sulphate groups before attachment of the dye.
SAPONIFICATION AS A MEANS OF SELECTIVELY REVERSING THE METHYLATION BLOCKADE OF TISSUE BASOPHILIA