Unlabeled Antibody Enzyme Research Articles

Immunohistochemical Demonstration of Pancreatic Secretory Proteins in Human Paneth Cells

Gastro-intestinal mucosa obtained at surgical resection was studied by light microscopy using the unlabelled antibody enzyme method for immunohistochemical staining of lysozyme, anionic trypsin, cationic trypsin and pancreatic trypsin inhibitor (PSTI). Paneth cells, identified by their content of lysozyme, contained anionic trypsin, cationic trypsin and PSTI-like immunoreactivity. The demonstration of immunoreactive trypsin and immunoreactive PSTI is a further indication of the resemblance between Paneth cells and the pancreatic acinar cells.

The coexistence of oxytocin and corticotropin-releasing factor in the hypothalamus: An immunocytochemical study in the rat, sheep and hedgehog

The unlabeled antibody enzyme method has been applied on adjacent sections in order to investigate coexistence of oxytocin (OXY) and corticotropin-releasing factor (CRF) within individual neurons of the hypothalamic paraventricular nucleus of the colchicine-treated rat, sheep and hedgehog. Our results show that, although OXY and CRF immunoreactivities are both expressed by a number of cells in the rat and the sheep paraventricular nucleus, this is not the case for the hedgehog.

Somatostatin-like immunoreactivity in non-pyramidal neurons of the human isocortex.

The distribution of somatostatin-immunoreactive cell bodies and axons throughout the human isocortex and subjacent white matter was examined. Vibratome sections of cortical tissue (30-40 micrometers thick) obtained at surgery were treated to reveal the antigen by the unlabelled antibody enzyme method. Two types of somatostatin-immunoreactive axons were present: short, coiled axons and extended ones that follow a straight course in various directions. Somatostatin immunoreactive nerve cell bodies were encountered in layers II-VI and in the subjacent white matter. The majority of labelled cells were found in the white matter and layer VI, and then in layers II and III. The immunoreactive perikarya were fusiform, triangular or multipolar in shape and did not show preferential orientation of their long axis. Frequently, the fusiform neurons in layer VI and in the white matter were aligned parallel to radiate bundles of myelinated fibres. The immunoreactive neurons gave rise to a few thick dendrites. Often thin axon-like processes could also be recognized, originating either from the cell body or from a thicker dendrite. After destaining of the chromogen and counterstaining with aldehydefuchsin and gallocyanin chromealum, the formerly immunoreactive neurons displayed a light and eccentrically located nucleus. The soma contained only a sparse amount of basophilic substance and was nearly devoid of lipofuscin granules. In electron micrographs, the cisterns of the rough endoplasmic reticulum (RER) were localized near the periphery of the soma. Immunoreactivity occurred along membranes of the RER cistern, outer mitochondrial membrane, and in particles 120-150 micrometers in diameter. Rounded areas (up to a diameter of 1 micrometer) lacked immunoreactivity. Furthermore, there were a few tiny lysosomes.

Immunocytochemistry of luteinizing hormone-releasing hormone and sexual maturation of the frog brain: Comparisons of juvenile and adult bullfrogs ( Rana catesbeiana)

An unlabeled antibody enzyme immunocytochemical procedure has been combined with an adjacent serial section approach to localize mammalian-like immunoreactive luteinizing hormone-releasing hormone (ir-LH-RH) in brains of juvenile and adult bullfrogs ( Rana catesbeiana). In juvenile animals modest specific immunocytochemical staining was found over cell bodies in the anterior preoptic area (aPOA) and in scattered neurosecretory endings in the outer layer of the median eminence (ME). In brains of adults, a comparatively robust staining was observed for perikarya in the medial septal nucleus, as well as in the aPOA; intense immunostaining of a well-differentiated ME also was noted for adults. Specific immunocytochemical staining was absent from other brain regions and in control preparations. Accordingly, changes both in the number and distribution of cells stained and in the quality of immunostaining were coincident with sexual maturation of the brain. This initial study of juvenile and adult bullfrogs suggests that ir-LH-RH in septal and preoptic areas may exert distinctly differing activities.

GAD-immunoreactive neural elements in the basilar pontine nuclei and nucleus reticularis tegmenti pontis of the rat. I. Light microscopic studies.

Immunocytochemical procedures employing the unlabeled antibody enzyme (PAP) method were used to visualize those neuronal elements in the basilar pontine nuclei (BPN) and nucleus reticularis tegmenti pontis (NRTP) of the rat which contain glutamic acid decarboxylase (GAD), a key enzyme involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA). Neuronal somata, axons, and axon terminals in the BPN and NRTP exhibited GAD-immunoreactivity. Both thick and thin varieties of labeled axons and terminals were distributed in varying densities throughout the BPN and NRTP. The greatest accumulation of labeled terminals was noted in the ventrolateral and lateral border regions of the BPN while a slightly less dense aggregation was observed along the ventral, ventromedial and midline regions of the pontine gray. Labeled fibers and terminals were also observed in the dorsal and ventral peduncular regions as well as central portions of the lateral, ventral, and medial pontine areas. Axonal and terminal labeling was present throughout the NRTP but no focal increases in density similar to those in the BPN were apparent. No obvious GAD-labeled fiber bundles could be observed to enter the BPN or NRTP. However, small fascicles of labeled axons were seen to course ventrally around the dorsolateral aspect of the cerebral peduncle to reach lateral pontine areas while other labeled axons descended through clefts in the mid-portion of the cerebral peduncle or passed through the medial lemniscus and around the medial portion of the cerebral peduncle to enter the pontine gray.

Immunohistochemical investigations of gut hormones in the colon of patients with Hirschsprung's disease

The distributions of gut hormones in the colon of Hirschsprung's disease were investigated by the peroxidase-antiperoxidase (PAP) immunohistochemical method. Three colonic segments (ganglionic, oligoganglionic, and aganglionic) were stained by the unlabeled antibody enzyme method. The immunoreactivity of vasoactive intestinal polypeptide (VIP) was found to be reduced in the oligoganglionic and aganglionic segments. Antisera to substance P and met-enkephalin demonstrated immunoreactive cells and fibers in the ganglionic segment, whereas these cells and fibers were almost completely absent in the oligoganglionic and aganglionic segments. A similar distribution was seen for the mucosal endocrine cells with somatostatin immunoreactivity. Antisera to neurotensin, motilin, bombesin, and cholecystokinin revealed no immunoreactivity in the normal colon or the three segments. The differences in these peptides between normal and impaired colonal segments may be one of the causes of colon constriction in Hirschsprung's disease.

Antisera to gamma-aminobutyric acid. I. Production and characterization using a new model system.

Antisera to the amino acid gamma-aminobutyric acid (GABA) have been developed with the aim of immunohistochemical visualization of neurons that use it as a neurotransmitter. GABA bound to bovine serum albumin was the immunogen. The reactivities of the sera to GABA and a variety of structurally related compounds were tested by coupling these compounds to nitrocellulose paper activated with polylysine and glutaraldehyde and incubating the paper with the unlabeled antibody enzyme method, thus simulating immunohistochemistry of tissue sections. The antisera did not react with L-glutamate, L-aspartate, D-aspartate, glycine, taurine, L-glutamine, L-lysine, L-threonine, L-alanine, alpha-aminobutyrate, beta-aminobutyrate, putrescine, or delta-aminolevulinate. There was cross-reaction with gamma-amino-beta-hydroxybutyrate, 1-10%, and the homologues of GABA: beta-alanine, 1-10%, delta-aminovalerate, approximately 10%, and epsilon-amino-caproate, approximately 10%. The antisera reacted slightly with the dipeptide gamma-aminobutyrylleucine, but not carnosine or homocarnosine. Immunostaining of GABA was completely abolished by adsorption of the sera to GABA coupled to polyacrylamide beads by glutaraldehyde. The immunohistochemical model is simple, amino acids and peptides are bound in the same way as in aldehyde-fixed tissue and, in contrast to radioimmunoassay, it uses an immunohistochemical detection system. This method has enabled us to define the high specificity of anti-GABA sera and to use them in some novel ways. The model should prove useful in assessing the specificity of other antisera.

Antisera to gamma-aminobutyric acid. III. Demonstration of GABA in Golgi-impregnated neurons and in conventional electron microscopic sections of cat striate cortex.

Two methods are described for the immunocytochemical demonstration of immunoreactive gamma-aminobutyric acid (GABA) in the visual cortex of the cat, an area that contains several types of GABAergic neurons and requires combined methods for their characterization. The first method is illustrated by a representative example of a Golgi-impregnated and gold-toned interneuron of the "bitufted" type situated in layer VI and having an ascending axon. After recording the three-dimensional features of the cell, semithin (0.5 micron) sections of the perikaryon were cut and GABA was demonstrated in the cell body by the unlabeled antibody enzyme method. While immunocytochemistry was used to determine the probable transmitter of the neuron, Golgi-impregnation of the same cell was used to identify its neuronal type. Since aldehyde-osmium fixation was used, further electron microscopic (EM) analysis of the neuron's synaptic connections was possible. The second procedure demonstrated GABA in EM sections of aldehyde-osmium-fixed cortex using protein A-gold as an immunocytochemical marker. Immunoreactivity was found in certain neurons, dendrites, axons, and boutons forming type II synaptic contacts that from previous studies have been thought to be GABAergic. Thus ultrastructural analysis using optimal conditions can now be supplemented with the identification of the transmitter in the same section.

Antisera to gamma-aminobutyric acid. II. Immunocytochemical application to the central nervous system.

An antiserum to gamma-aminobutyric acid (GABA) was tested for the localization of GABAergic neurons in the central nervous system using the unlabeled antibody enzyme method under pre- and postembedding conditions. GABA immunostaining was compared with glutamate decarboxylase (GAD) immunoreactivity in the cerebellar cortex and in normal and colchicine-injected neocortex and hippocampus of cat. The types, distribution, and proportion of neurons and nerve terminals stained with either sera showed good agreement in all areas. Colchicine treatment had little effect on the density of GABA-immunoreactive cells but increased the number of GAD-positive cells to the level of GABA-positive neurons in normal tissue. GABA immunoreactivity was abolished by solid phase adsorption to GABA and it was attenuated by adsorption to beta-alanine or gamma-amino-beta-hydroxybutyric acid, but without selective loss of immunostaining. Reactivity was not affected by adsorption to glutamate, aspartate, taurine, glycine, cholecystokinin, or bovine serum albumin. The concentration (0.05-2.5%) of glutaraldehyde in the fixative was not critical. The antiserum allows the demonstration of immunoreactive GABA in neurons containing other neuroactive substances; cholecystokinin and GABA immunoreactivities have been shown in the same neurons of the hippocampus. In conclusion, antisera to GABA are good markers for the localization of GABAergic neuronal circuits.

Primary neuroendocrine (Merkel cell?) carcinoma of the skin. II. An immunocytochemical study of 21 cases.

Twenty-one examples of neuroendocrine carcinoma of the skin were examined by the unlabeled antibody enzyme method for several neural hormones and peptides, carcinoembryonic antigen, S-100 protein, neuron-specific enolase, and three intermediate filaments: neurofilament, glial fibrillary acidic protein, and cytokeratin. Vasoactive intestinal polypeptide from two sources reacted with the neoplastic cells of four (18%) and seven (32%) of the cases, and pancreatic polypeptide reacted with scattered cells of one case. Neuron-specific enolase reactivity occurred in 50% of the cases. Neurofilament (70, 150, 200 kilodaltons) was strongly positive in 40% of the tumors whereas neurofilament (200 kilodaltons) was negative. Two monoclonal anticytokeratin antibodies of 54 kilodaltons and 44-54 kilodaltons reacted in 77% and 64% of the cases, respectively, in a distribution similar to the neurofilament. Sections reacted with antisera against cytokeratins of higher molecular weight were negative. The demonstration of vasoactive intestinal polypeptide, pancreatic polypeptide, neurofilament, and neuron-specific enolase is evidence of the neuroendocrine nature of this neoplasm.

The ultrastructural immunohistochemistry of oncofoetal antigens in large bowel carcinomas.

Seven large bowel carcinomas were examined by light and electron microscopy for the presence of five oncofoetal antigens. Ultrastructural investigations involved a novel method whereby thick sections of gluteraldehyde-fixed material were cut on a vibratome and then labelled using slight modifications of a standard unlabelled antibody-enzyme (PAP) technique, before further processing. Ultrastructural preservation, staining properties and the retention of antigen activity was seemingly better than that achieved by other investigators. Specific, positive labelling for carcinoembryonic antigen (CEA), colon specific antigen (CSA) and pregnancy-specific beta-1-glycoprotein (SP1) was seen in every case. Clear positive labelling for placental alkaline phosphatase (PLAP) and human chorionic gonadotropin (HCG) was seen in two cases. Extracellular labelling was found in areas of cell debris, free lying or in phagocytic cells and on tumour cell brush borders. The pattern of intracellular labelling, however, was different for each antigen and reflected the probable sites of synthesis and release from the cells. Thus CEA, a complex glycoprotein, was localised within the golgi apparatus, small apical cytoplasmic vesicles and mucous droplets in relatively well differentiated tumour cells. CSA, a chemically related glycoprotein, had a similar, but less dense distribution. SP1, by contrast, was localised within basally-located vesicles associated with the ribosomal endoplasmic reticulum and appeared to be released and persist as debris or taken up by phagocytic cells below the basal lamina. PLAP and HCG, both proteins, were found within simple single membrane-bound vesicles within relatively undifferentiated cells.

Different populations of GABAergic neurons in the visual cortex and hippocampus of cat contain somatostatin- or cholecystokinin- immunoreactive material

The coexistence of gamma-aminobutyric acid (GABA), glutamate decarboxylase (GAD), and cholecystokinin (CCK)- or somatostatin-immunoreactive material in the same neurons was studied in the hippocampus and visual cortex of the cat. One-micrometer-thick serial sections of the same neuron were reacted to reveal different antigens by the unlabeled antibody enzyme method. All CCK- and somatostatin-immunoreactive neurons in the cortex and all CCK-immunoreactive and the majority of somatostatin-immunoreactive neurons in the hippocampus that could be examined in serial sections were also immunoreactive for GABA. In neurons that were immunoreactive for GAD it was often possible to demonstrate immunoreactivity for one of the peptides as well as for GABA. GABA-immunoreactive neurons, as revealed by an antiserum to GABA, were present in all layers of the cortex and hippocampus, and their shape, size, and distribution were similar to GAD-immunoreactive neurons. All GAD-immunoreactive neurons were also positive for GABA, but the latter staining revealed additional neurons. CCK/GABA- and somatostatin/GABA-immunoreactive neurons were present mainly in layers II and upper III and in layers V and VI in the visual cortex. CCK/GABA-immunoreactive neurons were most frequently present in the strata oriens, pyramidale, and moleculare of the hippocampus and in the polymorph cell layer of the dentate gyrus. Somatostatin/GABA-immunoreactive neurons were localized mainly in the stratum oriens and in the hilus of the fascia dentata. The two peptides could not be found in the same neuron. The majority of neurons that were GABA immunoreactive did not stain for either peptide. The presence of CCK- and somatostatin-immunoreactive material in GABAergic cortical neurons raises the possibility that neuroactive peptides affect GABAergic neurotransmission.

Immunohistochemical investigations of leptospira-infected golden hamsters in potency tests

Golden hamsters taken from routine potency testing of leptospirosis vaccines were investigated for leptospirae after a challenge infection. Kidneys, livers, and spleens were examined immunohistochemically by the unlabelled antibody enzyme (PAP) method, and samples of these organs were additionally cultured for the evidence of living leptospirae. In non-vaccinated animals the recultivation method indicated leptospirae during the bacteraemic phase, beginning immediately after infection. Both methods resulted in positive reactions after organ manifestations. Intravascular evidence of leptospirae in the kidneys could be seen first on day 3 after infection. On day 7, there was intracellular and intratubu-lar evidence as well, combined with tubulo-interstitial alterations. In vaccinated animals the bacteraemia after infection was reduced. Organ manifestations were seen rarely; there were less severe or no pathohistological alterations.

Enrichment of lysozyme-containing cells from the rabbit endocervix by unit gravity sedimentation.

Lysozyme is a bacteriolytic enzyme component of the secretory granules of endocervical mucous cells. In order to study the subcellular distribution of this enzyme in specific cell populations, endocervical cells from estrous and 5-day pseudopregnant rabbits were separated by unit gravity sedimentation. The application of this technique to pronase-dispersed endocervical cells from estrous rabbits resulted in the isolation and enrichment of two mucous cell types that were distinguished morphologically into type I and type II cell populations. Lysozyme was identified in both cell types, using an unlabeled antibody enzyme method, and the degree of staining paralleled the number of mucous granules. In the absence of estrogen dominance in 5-day pseudopregnant rabbits, there was a 50% reduction in the number of mucous cells with a concomitant reduction in both the number of secretory granules per cell and the intracellular concentration of lysozyme. In the absence of ovarian steroid hormones, i.e., 15-16 weeks after ovariectomy, endocervical cells were devoid of secretory granules and lysozyme staining was negative. Enriched populations of endocervical cells represent a potential experimental model for studying the hormonal role in the regulation of lysozyme synthesis by specific cell populations.

Ultrastructural immunocytochemical localization of lysozyme in the mucociliary epithelium of the rabbit endocervix in different hormonal states.

The distribution of lysozyme in the endocervix of estrous, pseudopregnant, and ovariectomized rabbits was studied using two different immunocytochemical techniques--the unlabeled antibody enzyme method of Sternberger et al. (1970) and the peroxidase-labeled antibody method of Taylor and Burns (1974). With both procedures, a fine immunostaining precipitate was seen over the entire area of basal mucous granules, while immunodeposits were coarser and mostly located in the outer zone of central and apical granules. A nonspecific staining was noted when tissues were reacted with peroxidase-antiperoxidase complex alone. This troublesome artifact was abolished by preincubating tissues with human IgA. This step did not affect the specific immunostaining for lysozyme yet nonspecific staining was absent from specificity and method controls carried out for both immunocytochemical procedures. The presence of high levels of lysozyme in the endocervical epithelium of estrous rabbits was also confirmed in enzymatically isolated endocervical epithelia using the lysoplate method of Osserman and Lawlor (1966). Mucous granules and immunostainable intracellular lysozyme were abundant during estrus, decreased during early pseudopregnancy, and were absent after long-term ovariectomy. However, they were restored by the administration of estradiol (5 micrograms/12 hours/10 days) to ovariectomized animals. These data indicate a common hormonal regulation and secretory mechanism for endocervical mucous glycoproteins and lysozyme.

Immunomorphologic characterization of chicken thrombocytes.

Chicken thrombocytes were enriched for immunization by utilizing their strong capacity to adhere to plastic surfaces. The produced rabbit anti-thrombocyte serum ATS 3 reacted by means of the unlabeled antibody enzyme method (PAP) specifically with thrombocytes of fixed chicken-blood smears, but not with lymphocytes or other blood cells. When ATS 3 (substrate diaminobencidinetetra-hydrochloride = DAB) and a 1.1 mixture of an anti-bursa serum and anti-thymus serum (ABS/ATS; substrate 4-chloro-1-naphthol = 4-Cl-1-N) were used simultaneously, thrombocytes revealed the brown color typical for DAB, whereas lymphocytes showed the blue stain of 4-Cl-1-N. The finding of a thrombocyte surface antigen not shared by lymphocytes is regarded as a further proof of the diversity of both cell systems, i.e., for the existence of a genuine thrombocyte system in chickens.

Immunocytochemistry of luteinizing hormone-releasing hormone in brains of bullfrogs (Rana catesbeiana) during spontaneous metamorphosis.

The distribution of immunoreactive luteinizing hormone-releasing hormone (ir-LH-RH) in brains of bullfrogs (Rana catesbeiana) during spontaneous metamorphosis has been studied by combination of an unlabeled antibody enzyme immunocytochemical technique and an adjacent serial section approach. In prometamorphic tadpoles, immunocytochemical staining for ir-LH-RH was absent from the brain, including a structurally simple median eminence (ME) and perikarya in the anterior preoptic area (aPOA). In metamorphic tadpoles, speckled patches of immunostaining occurred over the outer layer of a modestly developed ME; coincident faint staining of a small number of medial, unpaired cell bodies was localized in the aPOA. In newly metamorphosed juvenile frogs, more diffuse and intense staining of the outer layer of the ME accompanied increased morphological differentiation of this neurohemal area; immunoreactive perikarya again were found in the aPOA, but an increased number of neurons exhibited comparatively greater (moderate) immunostaining. Changes in the quality of immunostaining and in the numbers of cells stained, therefore, were coincident with metamorphic development. Concomitant alterations of ir-LH-RH immunostaining and progressive structural development of the ME suggest a coordinated differentiation of brain neuroendocrine systems during metamorphosis of the bullfrog tadpole.

Stepwise amplified immunoperoxidase (PAP) staining. I. Cellular morphology in relation to membrane markers.

A novel procedure for the assay of monoclonal antibodies is described. The technique is based on a combination of three principles. Unlabeled (sheep) antiserum to mouse immunoglobulin (Ig) and complexes of peroxidase with mouse monoclonal antiperoxidase (monoclonal PAP complexes) are used as reagents in a variant of the unlabeled antibody enzyme (PAP) method, described by Sternberger. The amount of peroxidase eventually bound to a monoclonal antibody can be varied over a wide range by repetition of incubation cycles with anti-mouse Ig and monoclonal PAP complexes. During the assay, incubations and wash steps are performed by immersion of whole slides. The influence of repetitive incubation cycles with anti-mouse Ig and monoclonal PAP complexes on background staining and detection of monoclonal antibodies at low concentrations was quantitated in a model system. At a given primary antibody concentration, a linear relationship was found between peroxidase activity and the number of incubation cycles. Application of the technique to the detection of monoclonal antibodies bound to cell-surface antigens is described. Peripheral blood cells were labeled in suspension with monoclonal antibodies. Cytocentrifuge preparations of labeled cells were prepared, and such preparations were fixed before stepwise-amplified PAP staining. Cells showed intense specific staining. Morphological detail of stained and unstained cells is preserved, allowing morphological analysis of labeled cells and rapid analysis of monoclonal antibody specificity. Because the reagents used in the assay can be produced in large quantities with uniform quality, the technique can be readily automated. This, together with the possibility to increase the sensitivity of antibody detection in a controlled, stepwise fashion to levels that cannot be reached with "single-step" techniques, may further expand the applications of monoclonal antibodies.

Immunocytochemical evidence for the presence of enzymes synthesizing GABA and GHB in the same neuron

A specific and sensitive immunocytochemical double staining for visualization of glutamate decarboxylase (GAD) and semialdehyde succinate reductase (SSR 2) in the same brain section has been developed. SSR 2 is the enzyme responsible for the transformation of succinic semialdehyde into γ-hydroxybutyrate (GHB). GAD was detected using specific rabbit GAD-antibodies and unlabeled antibody enzyme peroxidase antiperoxidase, and SSR 2 using specific guinea-pig SSR 2 antibodies conjugate to a fluorescein-labeled second antibody. The coexistence of GAD and SSR 2 in the same neuron was demonstrated by a peroxidase reaction superimposed on fluorescent compounds. Cell bodies containing both antigens were observed in the cerebellum, dorso-median hypothalamus and raphe nuclei. GHB is present in most GABA containing neurons. Some neurons contain only SSR 2; these neurons may synthesize GHB by an active uptake of GABA.

Immunohistochemical evidence for the presence of γ-aminobutyric acid and serotonin in one nerve cell. A study on the raphe nuclei of the rat using antibodies to glutamate decarboxylase and serotonin

A specific and sensitive double immunocytochemical staining for the visualization of glutamate decarboxylase (GAD) and serotonin (5-HT) on the same brain section is developed. GAD is detected with specific GAD-antibodies by means of the unlabeled antibody enzyme, peroxidase anti-peroxidase, and serotonin with an antibody against the BSA-serotonin conjugate by an indirect immunofluorescent staining. The coexistence of GAD and 5-HT in the same perikaryon is demonstrated by a peroxidase reaction superimposed on fluorescent compounds. Cell bodies containing both antigens are observed in each raphe nuclei. However, the nucleus raphe dorsalis exhibits the largest number of cells containing either GAD alone or GAD and 5-HT together. An intracellular interaction between the metabolism of GABA and serotonin could be reasonably expected. The interactions between GABAergic and serotonergic systems must be thought of in terms of intracellular and/or transynaptic controls.