A microdensitometric method for the analysis of staining kinetics

The rate of histological staining is in general controlled by diffusion of dye in the dyebath and/or substrate. Microdensitometric and mathematical methods for studying the rate of staining are described, and illustrated by experiments on the staining of pancreatic cytoplasmic RNA and nuclei in sections by Azure at pH 4. Under given conditions the half-staining time, t1/2, of RNA was about twice that of nuclei, reflecting differences in permeability or porosity. Half-staining times were approximately doubled if the concentration of dye in the bath was reduced by 75%, suggesting that staining is not a simple first-order process; one possible explanation for this is that rate is affected by dye diffusion both in the dyebath and in the substrate. Lowering the temperature from 298 K to 277 K increased t1/2 for RNA about eightfold; the activation energy E of staining was therefore approximately 68 kJ mol-1. Assuming the substrate to consist either of plane sheets of thickness b, or uniform cylinders of radius r, equations originally proposed by Crank were applied to rate-of-dyeing data to calculate D/b2 and D/r2, where D is the diffusion coefficient of dye inside the substrate. From the observation that D/b2 and decreased markedly during the staining process it was concluded that both substrates studied were heterogeneous.