Quantitative cytochemical demonstration of intracellular thyroglobulin in cultured human thyrocytes. Effects of fixatives, TSH and interleukin-1β

A quantitative immunocytochemical method is described for measuring intracellular thyroglobulin in human thyrocytes grown in monolayer, based on the imidazole-enhanced 3,3'-diaminobenzidine/peroxidase reaction. The influence of ten different fixatives on the content of thyroglobulin immobilized on nitrocellulose filters and in single cells and the influence of thyrotropin and interleukin-1 beta (IL-1 beta) on the amount of intracellular thyroglobulin were evaluated. The most suitable fixatives for single cells were 2% carbodiimide, Lison's 'Gendre fluid' and 2 or 4% paraformaldehyde, whereas Bouin, Carnoy A and B, formalin-calcium and Lillie's formaldehyde-acetic acid-alcohol fixative all resulted in reduction of intracellular thyroglobulin. Two per cent glutaraldehyde caused a considerable reduction (p less than 0.0001). Nitrocellulose filters were not suitable for evaluation of the fixatives, since the results did not correspond to those obtained with single cells. Thyrotropin (1 U/l) increased intracellular thyroglobulin, whereas addition of interleukin-1 beta to the culture medium for three days caused a dose-dependent reduction with a plateau level at 2 x 10(-6) gl-1 (10(4) U/l) of interleukin-1 beta. It is concluded that changes in intracellular thyroglobulin concentration caused by either thyrotropin or IL-1 beta can be quantified under experimental circumstances where samples for measurements of thyroglobulin-mRNA or extracellular thyroglobulin are difficult or impossible to obtain.