Donald A. Keefer

Improved immunoglobulin-enzyme bridge method for light microscopic demonstration of hormone-containing cells of the rat adenohypophysis

SummaryAn improved procedure of high efficiency is described for light microscopic localization of tissue antigens by the unlabeled antibody-enzyme bridge technique with unpurified antiserum to horseradish peroxidase followed by horseradish peroxidase. The method incorporates blocking of nonspecific tissue binding sites with normal serum derived from the species in which the second component of the bridge was made, incubation for extended periods of time (42–65 hrs) with high dilutions (up to 1∶1,000,000) of the primary antibody, and optimal conditions for the histochemical reaction with diaminobenzidine and H2O2. Hormone-containing cells of the rat adenohypophysis can be stained with this technique using dilutions of the primary antisera that may approach, or exceed, those used in radio-immunoassay or in electron microscopic immunocytochemistry with preformed peroxidase-antiperoxidase complexes. Based partly on analysis of experimental data, it is suggested that the term “efficiency” should be used to describe the ability of immunohistochemical methods to work at high dilutions of the primary antisera, while the term “sensitivity” should be reserved to describe the ability of such methods to detect a specified amount or concentration of tissue antigen.

Quantitative autoradiographic assessment of 3H-estradiol uptake in immunocytochemically characterized pituitary cells

SummaryDry-mount autoradiography was combined with peroxidase immunocytochemistry to examine estrogen uptake in four pituitary cell types. Quantification by silver grain counts was used to compare 3H-estradiol uptake in nuclei of pituitary cells 60 min after i.v. injection into short-term (control) and long-term ovariectomized and in long-term thyroidectomized rats. Under all three hormonal states, the order of labeling intensity was: gonadotropes > somatotropes > lactotropes > thyrotropes. Long-term ovariectomy caused a significant increase in estrogen uptake of gonadotropes, somatotropes and lactotropes, while uptake in thyrotropes decreased. Long-term thyroidectomy decreased uptake in somatotropes, lactotropes and thyrotropes while gonadotropes remained unchanged.

Estrogen-Topographical Localization of Estrogen-Concentrating Cells in the Rat Spinal Cord Following 3H-Estradiol Administration

Summary After 3H-estradiol injection, radioactivity was found to concentrate in certain cells in the rat spinal cord. The topography was determined by dry-mount autoradiography. The greatest density at all levels occurred in laminae I and II of the dorsal horn. In the lumbar cord, a second group of labeled cells appeared in the dorsal portion of lamina X. Occasional scattered labeled cells appeared throughout the remaining gray and white matter. It is suggested that the radioactivity-concentrating cells are estradiol target sites involved in sex-related sensation and reflexes.

Estrogen-concentrating neuron systems in the brain of the tree shrew.

The distribution of radioactivity after the injection of [3H] estradiol-17β has been studied in the tree shrew (Tupaia glis), using dry-mount and thaw-mount autoradiography in conjunction with thin-layer chromatography. [3H] estradiol is concentrated and retained in nuclei of certain neurons which are accumulated in selective areas of the phylogenetically old periventricular brain, including rhinencephalic, limbic, and brainstem structures. An atlas of the topographic distribution of estrogen concentrating neurons is provided. The pattern of distribution in the preoptico-strial, basal hypothalamic, and amygdaloid region is similar to the pattern reported earlier for the rat, mouse, guinea pig, squirrel monkey, and ring dove.