What do you think you are quantifying? An appraisal of histochemical methods in the measurements of the activities of lysosomal enzymes.

The effect of a standard method of fixation (formalin-calcium 24 hr at 4 degrees C, followed by washing in gum-sucrose) on the activity of three lysosomal enzymes (N-acetyl-beta-D-glucosaminidase, acid phosphatase, beta-D-galactosidase) in the liver of three species (human, rabbit, lamb) was studied by biochemical methods, and the results were compared with staining intensities in histochemical preparations of the same tissues. The following conclusions were reached: (1) Fixation by formaldehyde changes the characteristics of the enzymes and makes comparisons between biochemistry and histochemistry difficult to interpret; (2) The intensity of staining in fixed or unfixed tissue sections bears no relation to absolute levels of enzyme activity; (3) Changes in staining intensity of a particular enzyme activity in a particular organ of a particular species prepared in a particular way are significant. Quantifying these changes is useful, as long as absolute values are not considered, and it is realized that it is only the difference that is being quantified.