Radiochemical determination of estrogen receptors in cryostat sections of target tissues.
A radioreceptor assay on unfixed cryostat sections has been developed. Mounted and dried sections were incubated with radiolabeled estradiol in the absence and presence of an excess of diethylstilbestrol and washed with buffer. Binding of radiolabel to sections was measured by direct liquid scintillation counting. Also protein-bound radioactivity which eluted from the sections was determined with a dextran-coated charcoal assay. Parallel sections were used for histological staining and protein determination. Scatchard analysis showed the presence of specific saturable binding sites for estradiol with dissociation constants in the 0.1-1.5 nM range. It is concluded that these high affinity and limited capacity (type I) binding sites represent estrogen receptors. The ligand-binding activity of section-bound estrogen receptors did not decrease upon dry storage up to 20 h at 4 or 23 degrees C prior to assay. During aqueous incubation a significant amount of receptor, representing about 40-60% of the total tissue content, elutes from the sections. Steroid specificity was proven by incubation with excess androgen, progestogen or corticoid instead of diethylstilbestrol or estradiol. With these ligands no significant competition was found. Tissue specificity was demonstrated by a very low level of specific estradiol binding to cryostat sections of rat skeletal muscle, spleen and intestine and by a moderate level in rat liver.
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D004958:Estradiol / Q000378:metabolism; D005260:Female; D005615:Freezing; D007700:Kinetics; D011869:Radioligand Assay / Q000379:methods; D011960:Receptors, Estrogen / Q000032:analysis* / Q000378:metabolism; D014316:Tritium; D014599:Uterus / Q000032:analysis* / Q000166:cytology
D011960:Receptors, Estrogen; D014316:Tritium; D004958:Estradiol