Unlabeled Antibody Enzyme Research Articles

An immunohistochemical study of hypophysis in Anguis fragilis (Reptilia, Anguidae)

Hypophysis morphology and immunocharacteristics of the adenohypophyseal cells in Anguis fragilis were described by light microscopy using conventional staining methods and an unlabeled antibody enzyme technique. The A. fragilis hypophysis showed a general morphology similar to that of other reptiles. The occurrence and distribution of cell types revealed somatotropic cells in the caudal pars distalis only as large cellular cords. Instead, prolactin cells were observed in the rostral and medial pars distalis, where they appeared isolated or clustered in small cellular cords. Melanotropic cells were confined to the pars intermedia in the same region of the corticotropic cells, which were also observed in the mediorostral pars distalis, where they appeared elongated in shape. Thyrotropic cells were detected in the pars distalis with a distribution similar to that of the gonadotropic cells, the former being globular but the latter variable in shape.

Involvement of lot cells in guidance of mitral cell axons

The olfactory marker protein, a protein specific to the olfactory sensory neurons, has been studied in mouse during embryogenesis and in the postnatal period up to 30 days, with the unlabeled antibody enzyme method of immunohistochemistry. Olfactory neurons, which are morphologically detectable in 10-day-old embryos, do not contain olfactory marker protein. The protein is present in the olfactory neuroepithelium at embryonic day 14 and its appearance coincides with the establishment of sensory synapses in the olfactory bulb. Neurons containing the protein increase in number up to 30 days after birth. At 15 days of embryonic life, immunostaining was observed in sensory axons at the rostral tip of the olfactory bulb, and by embryonic day 17 a plexus of stained fibres has covered the bulbar surface. Between embryonic day 15 and postnatal day 1, olfactory axons have been observed to reach the mitral cell layer. In the vomeronasal system the olfactory marker protein is present at later stages and both the receptors' perikarya and their axons and axon terminals in the accessory olfactory bulb show a lower level of staining than the olfactory system proper.This study of the olfactory marker protein has allowed us to correlate its appearance with significant developmental phenomena.

Immunohistochemistry and Tinctorial Affinity of Adenohypophysial Cells of the Rat SnakePtyas mucosus(Colubridae)

Immunocharacteristics of the adenohypophysial cells of the rat snakePtyas mucosus(Colubridae) were studied with an unlabeled antibody enzyme technique (PAP method) using rabbit antisera against mammalian/synthetic hypophysial hormones. Adenohypophysial cells were identified on the basis of their specific immunoreactivity with various heterologous antisera. As in other reptiles, there was regional localization of pars distalis (PD) cell types in this snake. The gonadotropic (GTH) cells were identified by their specific immunoreactivity with anti-porcine (p)LHβ serum and were distributed uniformly throughout the PD. The thyrotropic (TSH) cells, recognized by their specific immunoreactivity with anti-human (h)TSHβ serum, were found in the medial PD. The prolactin (PRL) and growth hormone (GH) cells were revealed by their specific immunoreactivity with anti-ovine (o)PRL and anti-hGH sera, respectively; the former were confined to the anterior two-thirds of the PD, and the latter were restricted to the posterior third of the PD. The corticotropic (ACTH) cells, identified by their specific immunoreactivity with the anti-ACTH1–24serum, were localized in the anterior two-thirds of the PD. Though both ACTH and PRL cells were confined to the same area of the PD, they could be distinguished by their distinctive morphology and distribution pattern. Cells of the pars intermedia were revealed by their immunoreactivity to anti-αMSH (melanophore-stimulating hormone) and anti-ACTH1–24sera. Among each adenohypophysial cell type, there was variation in the intensity of immunoreactivity and morphological features, which may be due to their heterogeneity, reflecting various stages of cellular activity. Unlike most other snake species, the occurrence of a rudimentary pars tuberalis inP. mucosuscontaining a few immunoreactive GTH and TSH cells appeared to be a novel finding. The occurrence of the PRL-like and TSH-like immunoreactivity seen in certain neuronal perikarya and fibers of the hypothalamus and median eminence conforms with earlier observations in other tetrapods. To resolve certain discrepancies in the literature, the tinctorial affinities of immunohistochemically identified adenohypophysial cell types ofP. mucosuswere studied using various conventional staining methods and were compared with those of other reptilian species studied earlier, including snakes of the family Colubridae.

Immunocytochemical localization of endogenous codeine and morphine

Experiments carried out by indirect immunofluorescence and unlabelled antibody enzyme procedures revealed the presence of morphine-like immunoreactive material in the perikarya, fibers, and terminals of neurons in different, discrete areas of rat and human brain. The monoclonal and polyclonal anti-morphine antibodies used do not distinguish between morphine and codeine. Endogenous morphine seems to be stored in neurons as the 3-ethereal sulphate conjugate. This possibility is supported by the finding that, although active uptake of [3H]morphine has not been detected in brain synaptosomes, long-term i.c.v. injection of the tritiated opiate results in the accumulation of radioactivity inside the same neurons in which the endogenous alkaloids have been detected. Finally, striatal slices exposed to high K+ concentrations showed a rapid disappearance of the morphine-like immunoreactive material from neurons, indicating that endogenous alkaloids are released from neurons by depolarization.

Immunohistochemistry of the pituitary pars distalis of the musk shrew, Suncus murinus

Immunocharacteristics of the pituitary pars distalis cell types of the musk shrew, Suncus murinus, were studied by the unlabeled antibody enzyme technique, using peroxidaseantiperoxidase or avidin-biotin-peroxidase complex. The thyrotropin (TSH)-, gonadotropin (GTH)-, corticotropin (ACTH)-, prolactin (PRL)-, and growth hormone (GH)-secreting cells of the PD were identified on the basis of their immunoreactivity with different heterologous antisera. The TSH cells showed specific immunoreactivity with antisera against human (h) TSHβ and rat (r) TSHβ. Cells showing immunoreactivity with the antisera against hLHβ and ovine (o) LHβ were designated as GTH cells as no immunoreactivity was observed with antisera against hFSHβ and oFSHβ. The ACTH cells as well as the cells of the pars intermedia were revealed by anti-ACTH 1–24 and anti-ACTH 1–10 sera. Whereas the PRL cells were recognized by their immunoreactivity with antisera against hPRL and oPRL, the GH cells were identified with anti-hGH, anti-oGH, and anti-bovine (b) GH sera. TSH and GTH, TSH and ACTH, GTH and ACTH, ACTH and GH, ACTH and PRL, and GH and PRL cells were visualized in the same section using the dual immunoperoxidase technique. Comparison of the immunohistochemically identified cells with those described histochemically reveals several discrepancies, which expose the limitations of the latter techniques identifying adenohypophysial cells.

Detection of estrogen receptor (ER) in the rat brain using rat anti-ER monoclonal IgG with the unlabeled antibody method

Application of Sternberger's unlabeled antibody enzyme method for detection of the estrogen receptor (ER) using a rat primary antibody with rat tissues has been discouraged, presumably because nonspecific staining of endogenous IgG was expected with the required anti-rat IgG bridging antibody. Because the blood-brain barrier greatly reduces immunoglobulin infiltration into the brain, we hypothesized that rat brain tissue could be specifically immunostained using rat IgG primary antibodies. A rat monoclonal anti-ER antibody (H222) specifically stained ERs in the brains of ovariectomized but not in ovariectomized estrogen-treated rats. In contrast, the uterus, a well-perfused target organ stained intensely in a nonspecific fashion. Dense populations of estrogen receptors were observed in the medial preoptic area, the bed nucleus of the stria terminalis, and the arcuate and ventromedial nuclei of the hypothalamus. A monoclonal rat IgG directed against alpha-tubulin labeled primarily cortical dendrites quite distinct from the neuronal nuclei that are the primary antigenic sites for the estrogen receptor antibody. These results confirm that the sensitive unlabeled antibody method can be applied to rat brain tissues, even when the primary antibody is rat IgG and that labeling of endogenous IgG may be used as a simple method to evaluate the integrity of the blood brain barrier.

Immunohistochemical demonstration of canine distemper virus antigen as an aid in the diagnosis of canine distemper encephalomyelitis

Brain tissue from 33 dogs with non-suppurative encephalitis was examined for evidence of canine distemper virus (CDV) encephalitis. Sections were examined for lesions, inclusion bodies, syncytial cells and CDV antigen using a double bridge unlabelled antibody enzyme technique. Histopathological lesions considered to be typical of granulomatous meningoencephalomyelitis were found in seven dogs. They all lacked inclusion bodies, syncytial cells and CDV antigen. The remaining 26 dogs all had histopathological lesions typical of CDV encephalitis. Inclusion bodies were found in 24 dogs, four of which also had syncytial cells and CDV antigen was detected immunocytochemically in 25. One dog had no inclusion bodies or syncytial cells and was immunohistochemically negative. Syncytial cells have been found to be of limited diagnostic value for the diagnosis of CDV encephalitis. While inclusion bodies proved to be a good diagnostic criterion for the confirmation of CDV infection, the immunohistochemical demonstration of CDV antigen proved to be superior. CDV antigen was more prevalent than inclusion bodies in tissue sections and much more easily detectable.

Neuropeptide Y-like immunoreactive neurons in the human olfactory bulb

Neuropeptide Y-like (NPY) immunoreactivity was localized in the adult human olfactory bulb by the unlabeled antibody enzyme (peroxidase anti-peroxidase; PAP) technique in vibratome sections. The majority of NPY-immunoreactive somata was localized in the white matter surrounding the anterior olfactory nucleus. Immunoreactive neurons were less numerous within the anterior olfactory nucleus and within the olfactory bulb layers. NPY-immunoreactive fibres were present in the white matter, the anterior olfactory nucleus, and in the olfactory bulb layers. Fibres within the white matter were generally aligned in a straight path parallel to the long axis of the olfactory bulb and tract. Fibres within the anterior olfactory nucleus showed no clear orientation and displayed numerous branching points. Coiled plexes of NPY-immunoreactive fibres were present in the glomerular layer of the olfactory bulb. Additional characteristics of the NPY-immunoreactive neurons were studied after decolouring the chromogen and restaining the sections with aldehydefuchsin to demonstrate the presence of lipofuscin granules and also with gallocyanin chrome alum to stain the Nissl substance. This analysis showed that the neurons belong to the class of non-pigmented nerve cells.

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the suprachiasmatic nucleus of the perinatal rat

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was examined in the suprachiasmatic nucleus (SCN) of the perinatal rat using the unlabeled antibody enzyme method of Sternberger. Our results showed that VIP-like immunoreactivity was present in cells and fibers of the SCN prior to birth. Immunoreactive neurons at this time appeared morphologically immature, and their distribution differed somewhat from that seen in the adult SCN.

VIP-like immunoreactivity in the anterior hypothalamic area of a hibernating bat varies with physiological state

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the anterior hypothalamic area (AHA) was studied in 15 bats which are seasonal hibernators, using the unlabeled antibody enzyme of Sternberger. Our results showed that the density of immunoreactive fibers and terminal plexuses in the AHA was greatest in euthermic animals and decreased dramatically when body temperature and cardiac rate were depressed and animals entered hibernation.

Immunohistochemical Identification of the Adenohypophysial Cells in the Indian Paddy Field Frog, Rana limnocharis

AbstractCells of the pituitary pars distalis (PD) and pars intermedia (PI) in the frog Rana limnocharis have been identified by an unlabelled antibody enzyme method using antisera developed in rabbit against mammalian hypophysial hormones. On the basis of their immunoreactivity, six types of cells, viz. thyrotropic (TSH), gonadotropic (GTH), prolactin (PRL), growth hormone (GH), corticotropic (ACTH) and melanotropic (MSH), cells have been recognized. GTH and PRL cells are distributed throughout the PD. GH cells usually occur in the anterodorsal and central region of the gland. Immunoreactive TSH cells are fewer in number and are localized in the ventromedian region of the PD. Cells showing immunoreactivity to ACTH 1–24 antiserum are encountered in the rostroventral part of the PD. Cells of the PI also show immunoreactivity to ACTH 1–24 antiserum. PI cells cross‐react with α‐MSH antiserum at all dilutions up to 1: 50 000. However, when the same antiserum was used at dilutions up to 1: 20 000, the ACTH cells of the PD also showed cross‐reactivity.

Ontogeny of the hypothalamo-neurohypophysial system in the toad, Bufo melanostictus: An immunohistochemical study

Employing unlabeled antibody enzyme technique, the development of the hypothalamo-neurohypophysial system of the common Indian toad, Bufo melanostictus, has been studied by using antiserum raised in rabbit against bovine neurophysin. Immunoreactivity to the antiserum is first observed at stage 22 ( Gosner, 1960) in the presumptive preoptic nucleus and at stage 23 in the presumptive median eminence and pars nervosa. As development proceeds, progressive increase in the immunoreactivity in all the components of the hypothalamo-neurohypophysial system is observed. The detection of neurohypophysial hormones (stages 22–23) much before the appearance of limb buds (stages 25–26) may be due to the greater sensitivity of the immunohistochemical technique.

Cytodifferentiation and immunocharacteristics of adenohypophysial cells in the toad, Bufo melanostictus

Employing the unlabelled antibody enzyme technique cytodifferentiation, immunocharacteristics and topographical distribution of melanotropic (MSH), adrenocorticotropic (ACTH), thyrotropic (TSH), prolactin (PRL), gonadotropic (GTH) and growth hormone (GH) secreting cells in the embryonic/larval as well as adult pituitary gland of the common Indian toad, Bufo melanostictus, have been studied by using antisera raised in rabbit against mammalian hypophysial hormones. Immunoreactive MSH and ACTH cells appear first in the dorsocaudal and rostral regions of the pituitary anlage (PA) at stage 21 (Gosner's classification) of the embryonic development. This is followed by the differentiation of TSH and PRL cells at stage 22 in the midventral and central regions of the PA respectively. Finally, at stage 23 the GTH cells appear in the rostral and the GH cells in the caudal regions of the PA. With the progress of the development, cells showing immunoreactivity to various antisera gradually increase in number, size, granular content and finally occupy the characteristic adult disposition. The MSH cells comprise the pars intermedia. In the pars distalis (PD) the ACTH cells are localized in the rostroventral region, TSH cells in the central region and the GH cells in the dorsocaudal region. However, GTH and PRL cells are distributed throughout the PD.

Glutamic acid decarboxylase immunoreactivity in sector CA1 of the human Ammon's horn.

The distribution of glutamic acid decarboxylase (GAD) immunoreactive neurons, fibres and punctae in sector CA 1 of the adult human Ammon's horn was studied in Vibratome sections (40 micron thick) of tissue obtained at surgery and autopsy. On light microscopical examination, the material did not show pathological changes. The antibody was visualized by the unlabelled antibody enzyme method. GAD-immunoreactive neurons, fibres and punctae were present in all layers. Most immunoreactive neurons were located in the stratum pyramidale and stratum lacunosum. Their size ranged from 8 microns in the stratum lacunosum to about 50 microns in the stratum oriens. The somata offered a wide range of shapes, multiform to fusiform with the long axis aligned parallel or vertically to the alveus. All somata belonged to the heterogeneous group of non-pyramidal neurons. The dendrites either radiated in all directions or tended to run in two opposite directions. After bleaching the chromogen and staining for lipofuscin pigment granules and basophilic material, it turned out that within the stratum pyramidale all formerly GAD-immunoreactive neurons belonged to the group of lipofuscin-laden non-pyramidal neurons. Within the other layers, a few formerly GAD-immunoreactive neurons were devoid of lipofuscin pigment. The highest density of GAD-immunoreactive punctae was found in the stratum lacunosum. In addition to numerous GAD-immunoreactive punctae in the pyramidal layer and in the stratum radiatum there were thin GAD-immunoreactive fibres of varying length extending into various directions.

GABA-immunoreactive neurons in the rat cerebellum: a light and electron microscope study.

An antibody raised against gamma - amino-butyric acid (GABA) coupled to bovine serum albumin with glutaraldehyde (Hodgson et al: J. Histochem. Cytochem. 33:229-239, '85) was used to localise immunocytochemically the presumptive GABAergic neuronal elements in the cerebellar cortex of the adult rat. employing the unlabelled antibody enzyme method with pre- and post-embedding immunocytochemical procedures, the following cellular structures were observed to be GABA-immunopositive in both the light and electron microscopes: the somata, dendrites, and axonal processes (including axon terminals) of stellate, basket, and Golgi neurons. In immunopositive neuronal somata and dendrites, the reaction product was found to be associated with all intracellular organelles and with the postsynaptic densities of synaptic junctions. Specific GABA-like immunoreactivity was also seen around outer mitochondrial membranes, microtubules, and neurofilaments, and coating synaptic vesicles in presynaptic axon terminals. In the pre-embedding procedure with dilutions of the antiserum between 1:1,000 and 1:2,000, the perikarya and dendrites of Purkinje cells were GABA-immunonegative, whereas at an antiserum dilution of 1:500 the somata of Purkinje cells were mildly GABA-immunoreactive. Purkinje cell axon terminals in the infra- and supraganglionic plexuses and in the deep cerebellar nuclei were always strongly immunopositive. Neuroglia were invariably GABA-immunonegative, as were the dendrites, axons (parallel fibres), and somata of granule cells. Mossy fibre and climbing fibre afferents were also immunonegative. The pattern of immunoreactivity obtained with this antiserum directed against the inhibitory neurotransmitter GABA was found to resemble closely the immunocytochemical distribution of GABA and of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) as reported previously in other immunocytochemical investigations (Oertel et al. and Wu et al: Cytochemical Methods in Neuroanatomy. New York: A. R. Liss, '82; Seguela et al: Neuroscience 16:865-874, '85; Mugnaini and Oertel: GABA and Neuropeptides in the CNS. Handbook of Chemical Neuroanatomy, Vol. 4, Part I. Amsterdam: Elsevier, '85.

Extraurophyseal distribution of urotensin II immunoreactive neuronal perikarya and their processes

The use of the unlabeled antibody enzyme method on serially adjacent sections permitted the demonstration of urotensin II (UII) and urotensin I (UI) immunoreactivities colocalized in most of the cells of the caudal neurosecretory system of Catostomus commersoni. The study of the upper regions of the central nervous system from the spinal cord anterior to the fifth preterminal vertebral region up to and including the brain stem revealed the presence of UII immunoreactivity in cerebrospinal fluid-contacting neurons, located ventral to the central canal along the entire length of the spinal cord and medulla. Beaded nerve fibers were observed projecting to the ventrolateral surface of the spinal cord and also forming a seemingly ascending immunoreactive-UII longitudinal bundle directed toward the brain. The presence of this "extraurophyseal" system of immunoreactive-UII cells and fibers suggests that the UII peptide may be released in upper regions of the central nervous system in response to stimuli conveyed via the cerebral spinal fluid. Thus, separate functions may be postulated for the urophyseal and the cerebral spinal fluid-contacting urotensin II systems.

Immunocytochemical localization of somatostatin in human brain

The distribution of somatostatin (SRIF) was examined in normal human forebrain, using thick vibratome cut sections. The unlabeled antibody enzyme method of immunocytochemistry revealed a widespread distribution of SRIF immunoreactive neurons and fibers throughout the septum, diencephalon and corpus striatum. Within the septum SRIF neurons and fibers were observed in the medial and lateral septal nuclei, the nucleus of the diagonal band, the nucleus accumbens and the bed nucleus of the stria terminalis. SRIF neurons and fibers were found in several hypothalamic and anterior thalamic nuclei as well as all regions of the corpus striatum. An interesting collection of SRIF immunoreactive neurons and processes were observed forming a wide band extending anteriorly from the lateral preoptic area through the lateral hypothalamus and substantia innominata posteriorly. This report on the localization of immunoreactive SRIF in the human forebrain extends previous anatomical findings and lends morphological support to recent biochemical studies.

Coexistence of immunoreactivity for melanin-concentrating hormone and α-melanocyte-stimulating hormone in the hypothalamus of the rat

Coexistence of immunoreactivity for melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH) within rat hypothalamic neurons has been examined by the unlabeled antibody enzyme method. Neurons exhibiting both MCH- and α-MSH-like immunoreactivities were found in the dorsolateral hypothalamus, whereas no MCH-like immunoreactive perikarya were seen in the arcuate nucleus, where some neurons were stained with α-MSH antiserum. There seem to be two distinct α-MSH-like immunoreactive neurons in the rat hypothalamus, one exhibiting coexistence with MCH-like immunoreactivity and the other not showing any cross-reaction with MCH antiserum.

Pancreatic endoproteases and pancreatic secretory trypsin inhibitor immunoreactivity in human Paneth cells.

Normal and metaplastic gastrointestinal mucosa obtained at surgical resection were studied by light microscopy, using the unlabelled antibody enzyme method for immunohistochemical staining of lysozyme, pancreatic endoproteases, and pancreatic secretory trypsin inhibitor (PSTI). Paneth cells in the mucosa of normal small intestine, gastric mucosa with intestinal metaplasia, and colonic metaplastic mucosa were found to contain anionic trypsin, cationic trypsin, lysozyme, and PSTI immunoreactivity, but not chymotrypsin and elastase immunoreactivity. Normal gastric and colonic mucosa and some goblet cells in the small intestine showed positive PSTI immunoreactivity but no endoprotease immunoreactivity. The presence of immunoreactive trypsin and immunoreactive PSTI in the Paneth cells, which are of secretory type, probably indicates an important extrapancreatic source of these proteins rather than a storage of endocytosed material.

Combined light and electron microscope immunocytochemical localization of scattered peptidergic neurons in the central nervous system.

A pre-embedding immunocytochemical technique is described for combined light and electron microscope study of peptidergic neurons in the central nervous system. The protocol is especially designed to overcome the sampling problems inherent in electron microscope study of structures, such as luteinizing hormone-releasing hormone (LHRH) neurons, that are scattered individually across large brain regions. The fixation methods outlined for several mammalian species include immersion and vascular perfusion with acrolein. Fine-structural preservation and LHRH immunoreactivity obtained with this fixative are compared to results with more conventional fixatives. Vibratome sectioning and a "pretreatment" regime, which prepare the tissues for immunocytochemistry, are described. Immunocytochemical labeling is done with free-floating sections and the peroxidase-antiperoxidase unlabeled antibody enzyme technique. Techniques are also described for the subsequent processing of immunoreacted sections for electron microscopy. These methods ensure that the processed sections are readily scanned by light microscopy, so that regions containing immunoreactive structures can be specifically chosen for electron microscope analysis. Sample electron micrographs are shown that illustrate some fine structural features of LHRH neurons in rats, bats, ferrets, and monkeys, as revealed with the techniques described.