L. S. Van Orden

A modification of the unlabeled antibody enzyme method using heterologous antisera for the light microscopic and ultrastructural localization of insulin, glucagon and growth hormone.

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.

On the use of the fluorescence histochemical method to estimate catecholamine content in brain

Abstract The relationship between intensity of formaldehyde-induced catecholamine fluorescence, determined microfluorematrically, and catecholamine content, measured biochemically, has been examined in variuos regions of rat brain. Linear relationships between these two parameters were obtained in the caudate nucleus, dorsomedial hypothalamus, arcuate nucleus, paraventricular nucleus, and internal and external layers of median eminence. Two pharmacological methods of catecholamine depletion were employed: inhibition of synthesis by α -methyl- p -tyrosine and interference with storage by reserpine. Fluorescence intensity and catecholamine content declined in a proportionate manner following either drug. The catecholamine content of single median eminence specimens was measured by an enzymatic-isotopic method. Catecholamine content of larger brain parts was measured by a fluorometric method. Both analytical methods employed could differentiate between norepinephrine and dopamine. The histochemical method did not differentiate between the two amines. It is concluded that microfluorometric evaluation of histochemical fluorescence intensity can be a useful and valid estimate of catecholamine content in certain regions of the brain.

Role of prostaglandins in estrogen-induced uterine hyperemia

Abstract Estrogen-induced uterine hyperemia is known to result from some other mechanism than direct vasodilatation produced by the hormone. Prostaglandin E 1 has been shown to exhibit properties consistent with a role as a vasodilator intermediate in estrogen-induced hyperemia. In the present study performed on rats the hyperemic response to estrogen as estimated by changes in uterine blood volume was blocked by two different prostaglandin synthesis inhibitors, indomethacin and meclofenamic acid. There was a parallel block of the increase in uterine PGE and PGF activity (radioimmunoassay) produced by estrogen. These results demonstrate that estrogen induces formation of uterine prostaglandins which appear to play a role in promoting the increase in blood supply to the uterus.

Serotonin in Hypothalamic Nuclei: Increased Content after Castration of Male Rats

Steady state levels of serotonin (5HT) in several hypothalamic nuclei of male rats increased when measured 7 days after castration. This effect was reversed by the administration of testosterone propi

Immunocytochemical localization of dopamine-β-hydroxylase in adrenal chromaffin granules

Abstract Electron microscopic localization of dopamine-β-hydroxylase (DBH) was carried out in bovine adrenal medullary chromaffin granule fractions and in intact bovine adrenal medulla by means of a peroxidase-antiperoxidase unlabelled antibody technique according to Sternberger , Hardy , Cuculis and Meyer (1970). Adrenal medullary chromaffin granule fractions showed retention of DBH despite successive fractionation, fixation and prolonged washing procedures, indicating that a portion of the DBH in the chromaffin granule is quite tightly bound. Under these circumstances the electron-opaque granular matrix was absent, implying loss of the more soluble constituents of the chromaffin granules, including matrix-associated DBH. Intact bovine adrenal medulla which was fixed, sectioned and subjected to immunocytochemical localization of DBH showed the enzyme to be present throughout the matrix of the chromaffin granule and demonstrated that, under these less vigorous conditions, the electron-opaque granule matrix was not removed. It would therefore appear to be feasible to differentiate directly between the “soluble” and “bound” forms of DBH by immunocytochemical procedures and to apply this technique to elucidate the process of exocytosis in adrenal medulla and adrenergic nerve terminals.