Unlabeled Antibody Peroxidase-antiperoxidase Method Research Articles

Ultrastructural-Immunocytochemical Localization of Growth Hormone and Prolactin in Human Pituitaries*

Immunocytochemical staining using the unlabeled antibody peroxidase-antiperoxidase method was undertaken to localize and characterize in ultrathin sections of human pituitaries the cells responsible for the secretion of GH and PRL. Somatotrophs in seven pituitaries stained with human (h) PRL-absorbed antiserum to hGH, were abundant, round to ovoid, densely granulated cells, whose mean (+/-SD) granule diameter was 368 +/- 60 nm. Lactotrophs immunostained with antiserum to hPRL were less numerous, angular or branching cells, with fewer round to ovoid granules, the mean diameter (+/-SD) of which was 185 +/- 35 nm in six pituitaries. The somewhat larger PRL granules (up to a mean diameter of 360 nm) seen in two of three additional pituitaries may have been related to the previous therapeutic administration of estrogen. Whereas the immunostained GH-secreting cells resemble the presumed somatotrophs identified in other studies on the basis of nonimmunological staining, the immunostained PRL-secreting cells differ considerably from the cells with large (600--1000 nm) granules designated as lactotrophs by several previous investigators. The hazards of ultrastructural identification of human pituitary cell types on purely morphological (as opposed to immunocytochemical) grounds are emphasized.

An immunohistochemical study of lysozyme in the human nasal mucosa.

The localization of lysozyme (LZM) in the human nasal mucoua was ob-served with the use of an unlabelled antibody peroxidase-antiperoxidase (PAP) method. LZM was distributed in serous cells of the nasal submucosal glands. Weak staining occurred in some mucous parts of them which seemed to be so-called seromucous cells classified by Shackleford and Wilborn, which exhibit intermediate characteristics between mucous and serous cells. In the present study, however, it was not clarified whether LZM produced by the serous cells is from blood plasma. No specific staining for LZM was detectable in the epithelial cells. Some tissue blood element cells such as neutrophils and macro-phages also contained antigenic LZM intracellularly in parallel with those of human blood huffy coats.

Immunohistochemical localization of adrenal ferredoxin and distribution of adrenal ferredoxin and cytochrome P-450 in the rat adrenal

Adrenal ferredoxin, the iron-sulfur protein associated with cytochromes P-450 in adrenocortical mitochondria, has been localized immunohistochemically at the light microscopic level in rat adrenals by employing rabbit antiserum to bovine adrenal ferredoxin in both an unlabeled antibody peroxidase-antiperoxidase method and an indirect fluorescent antibody method. When sections of rat adrenals were exposed to the adrenal ferredoxin antiserum in both procedures, positive staining for adrenal ferredoxin was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. Marked differences in the intensity of staining, however, where observed among the three cortical zones: the most intense staining being found in the zona fasciculata, less in the zona reticularis, and least in the zona glomerulosa. Furthermore, differences in staining intensity were also observed among cells within both the zona fasciculata and the zona reticularis. In agreement with these immunohistochemical observations, determinations of adrenal ferredoxin contents by electron paramagnetic resonance (EPR) spectrometry in homogenates prepared from capsular and decapsulated rat adrenals revealed that the concentration of adrenal ferredoxin in the zona glomerulosa was lower than that in the zona fasciculata-reticularis. Similar results were obtained when the contents of cytochrome P-450 were determined in capsular adn decapsulated rat adrenal homogenates. These observations indicate that adrenal ferrodoxin and cytochrome P-450 are not distributed uniformly throughout the rat adrenal cortex.

Hodgkin's disease: an immunohistochemical and histological study.

The distribution of immunoglobulin (Ig) in Hodgkin's tissue has been demonstrated in paraffin and cryostat sections by the unlabelled antibody peroxidase-antiperoxidase (PAP) method and the two-stage fluorescein isothiocyanate (FITC)-based technique. The morphology and histogenesis of the Reed-Sternberg (RS) and Hodgkin (HD) cells has been studied with the metalophil method of Marshall (1948) and this revealed a population of dendritic histiocytes which corresponded in number, sizes and distribution to the RS and HD cells in adjacent sections stained by haematoxylin and eosin. The largest RS cells, however, appeared to be non-dendritic. A notable feature of Hodgkin's tissue was the tendency for the dendritic cells to form "nodules", in combination with a population of small lymphocytes. The PAP technique reveals Ig in the form of mu and delta heavy chains, as well as kappa and lambda light chains, in close association with and probably on the surface of a high proportion of these lymphocytes, suggesting that they are immature B cells. Similar reactions were given by the mantle of lymphocytes of surviving normal lymphoid follicles, and metalophil dendritic histiocytes were also demonstrated within the mantle and subjacent part of the germinal centre. Numerous immunocytes containing Ig were present in all lesions; in the majority of cases, more cells contained gamma than alpha or mu heavy chains, although in these cases alpha-positive cells outnumbered those containing gamma or mu heavy chains. In two-thirds of the cases, one-third of the RS cells contained cytoplasmic immunoglobulin. This was exclusively gamma heavy, although both light chains were present. In about half the cases a minority of the HD cells contained gamma chain. The results suggest that HD and RS cells are dendritic histiocytes of the type normally found in the lymphoid follicles and that their tendency to form nodules in assoication with B lymphocytes is a manifestatin of this origin. It is suggested that the presence of Ig most probably results from absorption of antigen-antibody complexes on the cell surface.

Immunocytochemical detection of a murine leukemia virus-related nuclear antigen in mouse oocytes and early embryos

The expression of murine leukemia virus (MuLV)-related antigens in oocytes and early embryos of Swiss mice was studied by the unlabeled antibody peroxidase-antiperoxidase method using an antiserum to the major core protein, p30, of AKR MuLV. This procedure resulted in an intense staining, at antibody dilutions up to 1:500, of the germinal vesicles of oocytes and the interphase nuclei of early embryos. Nuclear staining was restricted to a specific period of oocyte growth and embryo development. It developed gradually in oocytes having reached one third to one half the full-grown size and persisted until meiotic maturation. In early embryos, nuclear staining was present from the one-cell stage to the morula, but disappeared during transition from morula to blastocyst and was not seen in expanded blastocysts. Nuclear staining was abolished by absorption of the anti-p30 serum with detergent-disrupted virions of Gross(A), AKR, Kirsten and Moloney MuLV, and Kirsten MSV(MuLV), but not with the Friend and Rauscher strains of MuLV, the xenotropic BALB:virus 2, a mouse tropic and an amphotropic clone of wild mouse MuLV, or with FeLV and RSV. On the basis of these results, it is suggested that the nuclear antigen (termed germinal vesicle antigen) reacting with the anti-p30 serum is the product of a cellular gene having a normal function in early embryonic development, and that sequences related to this gene are incorporated into the genome of AKR-type MuLV.

Immunohistochemical localization of adrenal ferredoxin in bovine adrenal cortex

Adrenal ferredoxin, the iron-sulfur protein associated with cytochrome P-450 in adrenocortical mitochondria, has been localized at the light microscopic level in bovine adrenal cortex. Localization was achieved through the use of rabbit antiserum to bovine adrenal ferrodoxin in an unlabeled antibody peroxidase-antiperoxidase method. When sections of bovine adrenal glands were exposed to the adrenal ferredoxin antiserum, intense staining was observed in parenchymal cells of the three cortical zones. Staining for adrenal ferredoxin was not detected in the medullary chromaffin cells. The presence of adrenal ferredoxin in the three cortical zones was verified by electron paramagnetic resonance spectrometry. These determinations also revealed that while the zona fasciculata and the zona reticularis contained approximately equal concentrations of adrenal ferredoxin, the concentration of the iron-sulfur protein in the zona glomerulosa was considerably lower. Similar results were obtained when the levels of cytochrome P-450 were determined in the three cortical zones. These results represent the first immunohistochemical localization within an intact tissue or cell of any component of an NADPH-dependent electron transport sequence which is responsible for the reduction of cytochrome P-450.

Electron microscopic immunocytochemical demonstration of separate mesotocinergic and vasotocinergic nerve fibres in the pars intermedia of the amphibian hypophysis

With the use of the unlabeled antibody peroxidase-antiperoxidase (PAP) method at the electron microscopic level, it has been shown that the pars intermedia of the hypophysis of Rana temporaria contains a diffuse intercellular network of separate mesotocinergic and vasotocinergic nerve fibres.

Immunohistochemical detection of growth hormone-release inhibiting hormone (somatostatin) in the guinea-pig brain.

Utilizing the unlabeled antibody peroxidase-antiperoxidase method, we have studied the distribution of somatostatin (growth hormone release-inhibiting hormone) in the guinea pig brain. An immunohistochemical reaction was found in both internal and external zone of the caudal and median portions of the median eminence. Reaction products were also present in the arcuate nucleus in proximity of the third ventricule. The immunostained elements resembled nerve processes. No cell bodies were stained. A positive reaction was also present in the whole organum vasculosum of the lamina terminalis. In the subfornical and subcommissural organs, somatostatin was observed in the ependymal and subependymal layers and the perivascular areas. In the pineal gland, a positive reaction was also found mainly in the perivascular areas. All the other brain areas were completely negative. These results establish that, in the hypothalamus, somatostatin is mainly concentrated in the median eminence and suggest that periventricular organs can play a role in the regulation of growth hormone secretion.

Growth hormone-release inhibiting hormone (GH-RIH) pathway of the rat hypothalamus revealed by the unlabeled antibody peroxidase-antiperoxidase method.

Utilizing the unlabeled antibody enzyme method, we investigated the distribution of hypothalamic elements immunoreactive with antibodies to growth hormone-release inhibiting hormone (GH-RIH). Immunostained elements, resembling neural processes, are distributed along a pathway corresponding to a portion of the tuberoinfundibular tract. However, GH-RIH fibers are caudal, dorsal and medial to LH-RH fibers detected by the same technique. Similar topographic arrangements are noted in coronal and sagittal sections. Comparable results were obtained with two different preparations of antisera to GH-RIH. No cell bodies specifically stained by anti-GH-RIH were detected. Our data agree with those of other investigators using immunohistochemical techniques.

Luteinizing hormone--releasing hormone (LH-RH) pathway of the rat hypothalamus revealed by the unlabeled antibody peroxidase-antiperoxidase method.

Utilizing the unlabeled antibody enzyme method, we report the distribution of hypothalamic elements immunoreactive with antibodies to luteinizing hormone-releasing hormone (LH-RH) in the rat. Immunostained elements, resembling neural processes, were distributed along a pathway corresponding to the tuberoinfundibular tract which appeared to terminate near vascular elements in the external layer of the preand post-infundibular median eminence. No cell bodies stained specifically for LH-RH. Similar topographic arrangements were noted (in coronal and sagittal sections) in diestrous females, ovariectomized females and a hypophysectomized male. The same results were obtained with three different preparations of antisera to LH-RH. Our studies agree with those of other investigators using immunohistochemical techniques as well as with localization studies of LH-RH in the hypothalamus using bioassay and radioimmunoassay. Our results suggest that the unlabeled antibody enzyme technique will have unique value for identifying and tracing fiber systems related to specific functions within the hypothalamus.

Identification of prolactin cells in a mammosomatotropic tumor by the unlabeled-antibody peroxidase-antiperoxidase method.

SummaryTo our knowledge, this is the first report on the immunocytochemical identification of prolactin-producing cells in the MtTW15 mammosomatotropic tumor variant. Cells staining positively for prolactin were characterized by extreme heterogenity of size, shape, and nuclear morphology. Only about 10% of the cells of tumor nodules were stained positively for prolactin. Roller cultures of MtTW15 tumor cells were not readily established; however, immunocytochemical staining revealed prolactin positive cells of diverse shapes in cultured cells as well.The authors express their gratitude to the NIAMDD for the rabbit anti-rat prolactin antiserum and to Ms. Karen Jensen and Virginia Lee for valuable technical assistance.