Immunohistochemical localization of glucagon-like peptide 1

Summary We report the use of poly-and monoclonal antibodies to study the immunohistochemical distribution of glucagon-like peptide-1 immunoreactivity (GLP-1-IR) in various tissues. The polyclonal antibodies against GLP-1 reacted with pancreatic A cells, enteroglucagon (L) cells in the gut, and some neurons in the central nervous system of all species tested. In pancreas and gut the monoclonal antibodies against GLP-1 exhibited a similar, but species specific distribution, relative to the polyclonal antibodies. The colocalization of GLP-1 and glucagon immunoreactivity in pancreatic, intestinal, and nervous tissues is in agreement with previously reported findings that both peptides are part of a single precursor molecule (preproglucagon).Supported by the DFG, SFB 90     

Detection of glucagon in pancreatic A-cells by monoclonal antibodies

The production of a mouse monoclonal antibody from a hybrid myeloma and its use for the detection of glucagon in tissue sections is reported. The hybrid clone isolated after fusion of mouse myeloma cells with hyperimmune spleen cells from a mouse previously immunized with porcine glucagon allowed us a standardized and permanent source of monoclonal antibodies in a culture cell system. The monoclonal antibody (3 GL 31) specifically reacts with pancreatic A-cells in several species including pig, rabbit, tupaia belangeri and sheep. No immunoreactivity is observed against gut cells and neurons.     

Detection of glucagon in pancreatic A-cells by monoclonal antibodies

Summary The production of a mouse monoclonal antibody from a hybrid myeloma and its use for the detection of glucagon in tissue sections is reported. The hybrid clone isolated after fusion of mouse myeloma cells with hyperimmune spleen cells from a mouse previously immunized with poreine glucagon allowed us a standardized and permanent source of monoclonal antibodies in a culture cell system. The monoclonal antibody (3 GL 31) specifically reacts with pancreatic A-cells in several species including pig, rabbit, tupaia belangeri and sheep. No immunoreactivity is observed against gut cells and neurons.Supported by the Deutsche Forschungsgemeinschaft, Carvas SFB 90     

Comparative immunohistochemistry and histochemistry of dipeptidyl peptidase IV in rat organs during development

Summary The occurrence of dipeptidyl peptidase (DPP) IV during development in Wistar rat organs was studied on day 10, 16 and 21 of gestation and on day 1, 4, 8, 13, 21, 26 and 60 after birth comparing immunohistochemistry and activity histochemistry. A polyclonal antibody, as well as monoclonal antibodies recognizing four different epitopes (A-D) of the DPP IV molecule, were employed for the immunohistochemical studies. In all investigated tissues, immunoreactivity with the polyclonal antibody appeared earlier than DPP IV activity and was already present on day 10 of gestation in the plasma membranes of embryonic and extraembryonic (decidual) cells. At these and other sites, e.g. brain capillary endothelium and tracheal or bronchial epithelium, immunoreactivity with the polyclonal antibody decreased or disappeared after birth and enzyme activity never developed. Immunoreactivity with the monoclonal antibodies appeared later than that with the polyclonal antibody, and mostly in those structures where DPP IV activity was subsequently found. The monoclonal antibody against epitope D showed a high reactivity in the epididymal duct, renal collecting ducts and in all domains of the hepatocyte plasma membrane, where neither DPP IV activity nor immunoreactivity with the other antibodies were observed. Our results also suggest that DPP IV might be present as a molecule before it becomes catalytically active and that immunoreactivity occurs at more sites than DPP IV activity. However, it cannot be excluded that the polyclonal antibody and the monoclonal antibody against the epitope D cross-react with as yet uncharacterized proteins, which express common epitopes during embryonic development, but are not present in the tissues of adult Wistar rats.     

Human specific anti-type IV collagen monoclonal antibodies, characterization and immunohistochemical application

This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryostat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution. Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal anti-type IV collage antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections. It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Human specific anti-type IV collagen monoclonal antibodies,characterization and immunohistochemical application

Summary This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryosat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution.Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal antitype IV collagen antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections.It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Localization of glucagon-like peptide (GLP) immunoreactants in human gut and pancreas using light and electron microscopic immunocytochemistry

The distribution of peptide immunoreactivities predicted from the sequence of the human preproglucagon gene in enteroglucagon (EG; glicentin-like immunoreactant-containing) cells of the human gut and A cells of the pancreas has been determined by light and electron microscopic immunocytochemistry. At light microscopy the application of peroxidase-antiperoxidase and immunogold-silver staining methods has revealed that glucagon-like peptide (GLP-1 and GLP-2) immunoreactivities coexist with a glicentin-related immunodeterminant in human colorectal EG cells and pancreatic A cells. Using single and double colloidal gold probe electron immunocytochemistry, we have been able to show the coexistence of glicentin, GLP-1, and GLP-2 immunoreactivities within single EG cell secretory granules. No morphologic segregation of the proglucagon immunoreactants was observed in EG cells of the colonic mucosa. In pancreatic A cells we have localized GLP-1, GLP-2, and glucagon-[16-29] immunoreactivities solely to the electron-dense core of the secretory granules, whereas glicentin-related immunoreactivity was restricted to the electron-lucent halo. The results obtained in the present study have shown that the peptide immunoreactivities predicted from cDNA sequencing of the human preproglucagon gene are indeed expressed in colorectal EG and pancreatic A cells. The topographical segregation of immunoreactivities in the A cell secretory granule shows that antigenic determinants derived from the C-terminal portion of proglucagon are stored with glucagon in the core of the secretory granule.     

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

Abstract. A panel of 10 mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFA) has been isolated using porcine GFA as antigen. Although all antibodies recognize GFA purified from porcine spinal cord in the western blot technique, they can be subdivided into at least three groups on the basis of their reactivity against defined fragments of the molecule. Immunofluorescence staining patterns with the monoclonal antibodies performed on tissues and cell lines resemble those reported with conventional polyclonal antibodies directed against GFA. In particular astrocytes and Bergmann glia are strongly stained. In addition mouse monoclonal antibodies specific for either the 200 kd, or the 160 kd, or the 68 kd neurofilament triplet protein have been isolated and characterized. These antibodies are specific for neuronal cells and support conclusions made with similar antigen affinity-purified polyclonal antibodies. The combined set of monoclonal antibodies seems a valuable tool to characterize the different cell types of the nervous system.     

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

A panel of 10 mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFA) has been isolated using porcine GFA as antigen. Although all antibodies recognize GFA purified from porcine spinal cord in the western blot technique, they can be subdivided into at least three groups on the basis of their reactivity against defined fragments of the molecule. Immunofluorescence staining patterns with the monoclonal antibodies performed on tissues and cell lines resemble those reported with conventional polyclonal antibodies directed against GFA. In particular astrocytes and Bergmann glia are strongly stained. In addition mouse monoclonal antibodies specific for either the 200 kd, or the 160 kd, or the 68 kd neurofilament triplet protein have been isolated and characterized. These antibodies are specific for neuronal cells and support conclusions made with similar antigen affinity-purified polyclonal antibodies. The combined set of monoclonal antibodies seems a valuable tool to characterize the different cell types of the nervous system.     

Cytochrome-C localizes in secretory granules in pancreas and anterior pituitary

We used quantitative immunogold electron microscopy to evaluate the subcellular distribution of cytochrome-c in normal rat tissues, employing a wide variety of monoclonal and polyclonal antibodies against mammalian cytochrome-c. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows highly specific labeling of mitochondria in all tissues examined, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen and thyroid. In pancreatic acinar cells and anterior pituitary, however, there was also strong cytochrome-c reactivity in zymogen granules and growth hormone granules, respectively. In the pancreas, strong immunoreactivity is also detected in condensing vacuoles and in the acinar lumen. Immunocytochemical controls included (i) use of monoclonal antibodies to horse cytochrome-c which recognize an epitope not present in rat cytochrome-c, (ii) preadsorption of antibodies with purified cytochrome-c, and (iii) omission of the primary antibody. The indicated presence of cytochrome-c outside mitochondria in certain tissues under normal physiological conditions raises interesting questions concerning translocation mechanisms and the cellular functions of cytochrome-c.     

Light and electron microscopical immunocytochemistry of 5'-nucleotidase in rat cerebellum

5'-Nucleotidase in nervous tissue has so far not been localised at the ultrastructural level using immunocytochemical techniques. We have now applied monoclonal antibodies and a polyclonal antiserum raised against this ecto-enzyme and describe the distribution of 5'-nucleotidase antigenicity in rat cerebellum both at the light and electron microscopic levels. Within all cerebellar layers, 5'-nucleotidase immunoreactivity was found on plasma membranes of glial elements, i.e. Bergmann glial cell processes crossing the molecular layer, astrocytic end-feet around blood vessels and glial cell extensions surrounding single Purkinje cells. In the granular layer, 5'-nucleotidase immunoreactivity was present on glial membranes interposed between granule cells. Neuronal cells or processes were devoid of immunoreactivity. The immunocytochemical results were compared with conventional 5'-nucleotidase histochemistry. Both techniques showed the same ecto-localisation of the enzyme and favour the view of 5'-nucleotidase being predominantly situated at glial plasma membranes.     

Immunodetection of secretogranin II in animal and human tissues by new monoclonal antibodies.

Secretogranin II (chromogranin C) is an acidic tyrosine-sulfated secretory protein, known to be a marker of neuroendocrine secretory products and of specific neuroendocrine tumours. In order to obtain anti-secretogranin II monoclonal antibodies for cell biology studies and, in particular, for clinical applications, we immunized mice with a secretogranin II-enriched fraction prepared from homogenates of bovine anterior pituitaries. Hybridoma supernatants obtained from the splenocytes of a hyperimmune mouse, screened with an enzyme-linked immunosorbent assay, were analyzed by both immunocytochemistry and two-dimensional immunoblotting. By using this experimental approach, we were able to identify two monoclonal antibodies (8G1 and 5A7) which recognize bovine secretogranin II. Both immunocytochemistry and immunoblotting revealed that one of them, the 5A7 antibody, cross-reacts with the human antigen. The distribution patterns of the immunoreactivity, obtained by immunocytochemistry with the 5A7 antibody in animal and human tissues, partially overlap those, obtained by using a polyclonal antiserum elicited against bovine secretogranin II, previously described. Moreover, the 5A7, but not the polyclonal antibody, reacts with some duodeno-jejunal cells. In conclusion, both the 5A7 and 8G1 antibodies can be useful for cell biology studies. The 5A7 antibody can be used for the detection of secretogranin II in human tissues and should be of help in clinical and pathological practices.     

Chromogranin A in the pancreatic islet: cellular and subcellular distribution

Chromogranin A (CGA) is the major soluble protein within secretory vesicles of chromaffin cells. A polyclonal antiserum was raised against bovine CGA and characterized in two-dimensional immunoblots. Cellular and subcellular distribution of CGA in bovine pancreatic islet was investigated by immunocytochemistry. At the light microscopic level, CGA-like immunoreactivity was found in the same cells that react with antibodies against insulin, glucagon, and somatostatin. A minority of cells containing pancreatic polypeptide also showed faint immunostaining. At the ultrastructural level (protein A-gold technique), CGA-like immunoreactivity was confined exclusively to the secretory vesicles. Whereas the hormones were localized mainly in the central part of the secretory vesicles, CGA was present predominantly in the periphery. These findings indicate that a CGA-like protein is a regular constituent of the matrix of secretory vesicles in pancreatic endocrine cells.     

Specificity of the localization of transforming growth factor-alpha immunoreactivity in colon mucosa.

Transforming growth factor-alpha (TGF-alpha) plays an important role in gastrointestinal pathophysiology. However, the exact location of its expression in the intestine is still controversial. This study systematically compared the localization of TGF-alpha immunoreactivity in frozen or fixed human colon using three different antibodies and examined specificity of antibodies by using tissues from TGF-alpha knockout mice and by Western blotting. Consistent with the mRNA distribution revealed by in situ hybridization, a similar staining pattern was obtained in frozen sections by all three antibodies, localizing on the surface and along the crypt epithelium. In paraffin sections, although the polyclonal antibodies (raised against recombinant human or rat TGF-alpha) gave minimal staining, the monoclonal antibody (against C-terminal peptide of human TGF-alpha) still gave intense staining on the surface and upper crypt epithelium. By using specimens from TGF-alpha knockout mice in immunostaining and Western blotting, the polyclonal antibodies were shown to be specific. In contrast, specificity of the monoclonal antibody was in doubt in rodent tissues because it gave similar detection between wild-type and knockout mice in both analyses, indicating its crossreaction to non-TGF-alpha molecules. In conclusion, frozen sections and antibodies raised from recombinant TGF-alpha should be used for TGF-alpha immunohistochemistry in the colon.     

Transthyretin immunoreactivity in human and porcine liver, choroid plexus, and pancreatic islets

We examined transthyretin immunoreactivity (TTR-IR) in human and porcine liver, choroid plexus, and pancreatic islets with both polyclonal and monoclonal antibodies to TTR. The specificity of the immunoreactions and the effects of various fixatives were tested in immunohistochemical and dot-blot systems. B-5 fixative (mercuric chloride and sodium acetate in formalin) was the best immunopreservative. In both species, the TTR-IR in choroid plexus epithelial cells was strong and was much greater than that in hepatocytes. Glucagon cells in pancreatic islets were also strongly TTR immunoreactive. Insulin cells were slightly TTR immunoreactive in human but strongly so in porcine pancreas. The finding of TTR-IR in normal islets explains the presence of TTR-IR in human endocrine pancreatic tumors, notably glucagonomas and malignant insulinomas.     

Trk-neurotrophin receptor-like immunoreactivity in the gut of teleost species

Neurotrophins, acting through their high-affinity signal-transducing Trk receptors, are involved in the development, differentiation and maintenance of discrete neuron populations in the higher vertebrates. Furthermore, the presence of Trk receptors in some non-neuronal tissues, including the endocrine cells of the gut, could indicate an involvement of neurotrophins also in these tissues. Recently, neurotrophins and neurotrophin receptor proteins have been identified in the lower vertebrates and invertebrates, whose amino acid sequences are highly homologous with those found in mammals. The present study investigates the occurrence and distribution of Trk-like proteins in the neurons and gut endocrine cells in five species of teleost. Single and double immunolabeling was carried out on fresh and paraffin-embedded tissue using commercially available antibodies against sequences of the intracytoplasmic domain of the mammalian Trk. Western-blot analysis, carried out on samples of stomach and intestine of bass, identified proteins whose estimated molecular masses (140 kDa, 145 kDa and 143–145 kDa) were similar to those reported for full-length TrkA, TrkB and TrkC in the higher vertebrates. TrkA-like immunoreactivity was found in the enteric nervous system plexuses of three fish species. Trk-like immunoreactivity was observed in the endocrine cells as follows: sparse TrkA-like immunoreactive endocrine cells were detected only in the intestine; TrkB-like immunoreactive cells were detected only in the stomach; and TrkC-like immunoreactive cells were found both in the intestine of the carp and in the stomach of the bass, where they also showed TrkB-like immunoreactivity. These findings confirm the occurrence and distribution of Trk-like proteins in teleosts. These proteins are closely related to the Trk neurotrophin receptors of mammals. The functional significance of Trk-like proteins in both neuronal and non-neuronal cells of teleosts is still not clear. Received: 3 August 1998 / Accepted: 2 October 1998     

Monoclonal Antibodies to Mammalian Carnosine Synthetase

A set of mouse monoclonal antibodies has been generated against rabbit muscle carnosine synthetase. The immunoreactivity of these antibodies has been characterized using an immunoassay that permits the separation and direct measurement of the synthetase activity on a second antibody bead complex. Four IgG monoclonal antibodies bind the carnosine synthetase activity from muscle of all mammals tested (mouse, rat, rabbit, cow, dog, and monkey) but not that from chicken muscle. This indicates the mammalian enzymes share epitopes that are absent from the avian enzyme. In addition, relative tissue levels of synthetase activity can be quantified with this immunoassay. Thus, high levels of carnosine synthetase activity are immunoprecipitated from the olfactory tissues of both rat and rabbit. Synthetase activity is generally lower in other tissues (muscle, brain, heart, liver, and gut). Nevertheless, the cross-reactivity of the synthetase from several tissues (olfactory mucosa, muscle, brain, gut, heart, and liver) of a single species indicates the enzyme protein contains similar epitopes in these tissues. Immunoaffinity purification of this low-abundance, unstable enzyme should now be possible for subsequent studies of structure and regulation.     

Distribution of tau proteins in the normal human central and peripheral nervous system

In human brain, antibodies to tau proteins primarily label abnormal rather than normal structures. This might reflect altered immunoreactivity owing to post-mortem proteolysis, disease, or species differences. We addressed this issue by comparing the distribution of tau in bovine and human post-mortem nervous system tissues and in human neural cell lines, using new monoclonal antibodies (MAb) specific for phosphate-independent epitopes in bovine and human tau. In neocortex, hippocampus, and cerebellum, immunoreactive tau was widely expressed but segregated into the axon-neuropil domain of neurons. In spinal cord and peripheral nervous system, tau immunoreactivity was similarly segregated but less abundant. No immunoreactive tau was detected with our MAb in glial cells or in human neural cell lines that express neurofilament or glial filament proteins. Post-mortem delays in tissue denaturation of less than 24 hr did not affect the distribution of tau, but the method used to denature tissues did, i.e., microwave treatment preserved tau immunoreactivity more effectively than chemical fixatives such as Bouin's solution, and formalin-fixed tissue samples reacted poorly with our anti-tau MAb. We conclude that the distribution of tau proteins in human nervous system is similar to that described in perfusion-fixed experimental animals, and that visualization of normal immunoreactive tau in human tissues is critically dependent on the procedures used to denature post-mortem tissue samples. Furthermore, microenvironmental factors in different neuroanatomical sites may affect the regional expression of tau.     

Distribution of anti-Leu-7, anti-Leu-11a and anti-Leu-M1 immunoreactivity in the brain of the adult rat

Summary This study reports a specific cross-reactivity of the three anti-human-hematopoetic-cell monoclonal antibodies, anti-Leu-7 (HNK-1), anti-Leu-11a (NKP-15), and anti-Leu-M1 (MMA), with different epitopes in the brain of the adult rat. The distribution of these epitopes in rat brain is determined by means of immunohistochemistry in paraffin-embedded frontal serial sections.The reaction pattern of anti-Leu-11a monoclonal antibody is very similar to that of polyclonal antibodies against the myelin basic protein. Both antisera give a specific reaction with myelinated fibers. Immunoreaction products with the anti-Leu-7 monoclonal antibody are found as diffuse, mostly punctiform material in the neuropil and even more evident as small granules coating the cell surface of many neurons. In the white matter anti-Leu-7 reveals a moderate reactivity, which occurs predominantly as spots and fine-stranded material within the myelinated fiber tracts.Anti-Leu-M1 immunoreactivity is present between myelinated fiber bundles of the white matter, where it has a reticulate appearance, and as fine-granulated material within the grey matter of the cortex and the nuclei. The characteristic feature in the grey matter is that of irregularly shaped immunopositive plaques, which are often located around small blood vessels. The cytoplasm of glial and neuronal cells appeared negative with this MAB.The exact topographical distribution of the Leu-7 and Leu-M1 epitopes throughout the rat brain is described. The present hypotheses concerning the nature of this shared antigenicity between hematopoetic cells and nervous tissue are discussed.Supported by the Deutsche Forschungsgemeinschaft, SFB 200     

Monoclonal antibodies to laminin reveal the heterogeneity of basement membranes in the developing and adult mouse tissues

Two monoclonal antibodies raised against laminin isolated from a mouse parietal yolk sac cell line were used for immunohistochemical studies of basement membranes of the mouse embryo and various fetal and adult tissues. No immunoreactivity with either of the two monoclonal antibodies could be detected in the preimplantation-stage embryos, although it has been shown that these embryos contain extracellular laminin reactive with the conventional polyclonal antilaminin antibodies. Reichert's membrane in early postimplantation stages of development reacted with the monoclonal antibody LAM-I but not with the antibody LAM-II. However, from day 8 of pregnancy onward the Reichert's membrane reacted with both antibodies. Basement membranes of the embryo proper were unreactive with both monoclonal antibodies until day 12 of pregnancy. By day 14 some basement membranes of the fetal tissues became reactive with one or both monoclonal antibodies, whereas others remained still unreactive. In the 17-d fetus and the newborn mouse most of the basement membranes reacted with both monoclonal antibodies, whereas others still reacted with only one. Similar heterogeneity in the immunoreactivity of basement membranes of various tissues was noted in the adult mouse as well. These results indicate that the immunoreactivity of laminin in the extracellular matrix changes during development and that the basement membranes in various anatomic locations display heterogeneity even in the adult mouse.