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

Abstract

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.

Relationship between immunostaining intensity and antigen content in sections.

“…Recently, video image processors and microphotometers have been used to quantitate the staining intensity of enzyme immunohistochemical reactions (Akner et al, 1994;Alford et al, 1994;Coventry et al, 1994;Verschure et al, 1994;Watanabe et al, 1991Watanabe et al, ,1993Watanabe et al, ,1994Dodge et al, 1993;Shimid et al, 1993;Carter et al, 1992;Dobado-Berrios et al, 1992;Kayser et al, 1991). When immunostaining intensity in a section is proportional to amount of an antigen in the section, antigen content in the section can be measured by quantitative immunohistochemical methods (Dobado-Berrios et al, 1992;Watanabe et al, 1991;Chu et al, 1989;Nibbering and van Furth, 1987).…”

A Simple Way to Improve Immunoreactivity of Prostate Needle Biopsies Fixed in Bouin’s Solution to AMACR

“…Some works that compared immunoreactivity of tissues fixed in formalin versus those fixed in Bouin's solution show different, and often contrary, results for different antibodies. [9][10][11][12] In the case of AMACR and Bouin's solution, we and others have seen that the antibody works, although some authors have discouraged the use of this fixative for prostatic biopsies if immunohistochemical analysis is planned, as there are very limited data available regarding their applicability to specimens processed in fixatives other than formalin. 12 In our experience, immunoreactivity, effectively, is decreased compared with that of the formaldehyde-fixed sections.…”