Quantitative cytochemical demonstration of intracellular thyroglobulin in cultured human thyrocytes. Effects of fixatives, TSH and interleukin-1 beta.
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.
Kayser
L
L
Department of Medical Anatomy A, Panum Institute, University of Copenhagen, Denmark.
Olsen
B E
BE
Høyer
P E
PE
D002478:Cells, Cultured; D005404:Fixatives / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D007375:Interleukin-1 / Q000494:pharmacology*; D013954:Thyroglobulin / Q000032:analysis*; D013961:Thyroid Gland / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D013972:Thyrotropin / Q000494:pharmacology*
D005404:Fixatives; D007375:Interleukin-1; D013972:Thyrotropin; D013954:Thyroglobulin