Synaptic Vesicle Protein 2, A New Neuroendocrine Cell Marker
2000; Elsevier BV; Volume: 157; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)64645-7
ISSN1525-2191
AutoresGuida Maria Portela‐Gomes, Agneta Lukinius, Lars Grimelius,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoSynaptic vesicle protein 2 (SV2) is a glycoprotein identified in the nervous system of several species, including man, but its occurrence in the human neuroendocrine (NE) cell system has not been investigated. By using a monoclonal antibody to SV2, immunoreactivities were demonstrated in NE cell types in human gastrointestinal tract, pancreas, anterior pituitary gland, thyroid, parathyroid, and adrenal medulla, and also in chief cells of gastric oxyntic mucosa. Immunoelectron microscopy of pancreatic islets revealed SV2 immunoreactivity in secretory granules. Comparison of SV2, synaptophysin, and chromogranin A immunoreactivity showed more SV2- and synaptophysin- than chromogranin A-immunoreactive cells in the antrum and pancreas. In the other gastrointestinal regions and in the other endocrine organs more SV2- than synaptophysin-immunoreactive cells were seen. More chromogranin A- than SV2-immunoreactive cells were observed in duodenum, colon, and parathyroid. Various NE tumors were examined and all contained SV2-immunoreactive cells. The staining patterns with the three markers agreed well, except in hindgut carcinoids, which showed strong SV2 immunoreactivity, weak synaptophysin but no chromogranin A immunostaining. In pituitary adenomas more cells were immunoreactive to SV2 than to the other two antibodies. In conclusion, SV2 is recognized as a further broad marker for NE cells and widens the arsenal of diagnostic tools for NE tumors. It is of special importance for identifying hindgut carcinoids. Synaptic vesicle protein 2 (SV2) is a glycoprotein identified in the nervous system of several species, including man, but its occurrence in the human neuroendocrine (NE) cell system has not been investigated. By using a monoclonal antibody to SV2, immunoreactivities were demonstrated in NE cell types in human gastrointestinal tract, pancreas, anterior pituitary gland, thyroid, parathyroid, and adrenal medulla, and also in chief cells of gastric oxyntic mucosa. Immunoelectron microscopy of pancreatic islets revealed SV2 immunoreactivity in secretory granules. Comparison of SV2, synaptophysin, and chromogranin A immunoreactivity showed more SV2- and synaptophysin- than chromogranin A-immunoreactive cells in the antrum and pancreas. In the other gastrointestinal regions and in the other endocrine organs more SV2- than synaptophysin-immunoreactive cells were seen. More chromogranin A- than SV2-immunoreactive cells were observed in duodenum, colon, and parathyroid. Various NE tumors were examined and all contained SV2-immunoreactive cells. The staining patterns with the three markers agreed well, except in hindgut carcinoids, which showed strong SV2 immunoreactivity, weak synaptophysin but no chromogranin A immunostaining. In pituitary adenomas more cells were immunoreactive to SV2 than to the other two antibodies. In conclusion, SV2 is recognized as a further broad marker for NE cells and widens the arsenal of diagnostic tools for NE tumors. It is of special importance for identifying hindgut carcinoids. Neuroendocrine (NE) cells contain two types of vesicular structures, small clear synaptic vesicles and large electron dense secretory granules. Several proteins have been observed in these vesicular structures, and among them chromogranin A and synaptophysin have attracted great interest. Chromogranin A occurs in most NE cell types,1Lloyd RV Wilson BS Specific endocrine tissue marker defined by a monoclonal antibody.Science. 1983; 222: 628-630Crossref PubMed Scopus (293) Google Scholar, 2Portela-Gomes GM Stridsberg M Johansson H Grimelius L Complex co-localization of chromogranins and neurohormones in the human gastrointestinal tract.J Histochem Cytochem. 1997; 45: 815-822Crossref PubMed Scopus (71) Google Scholar and has been used during the last two decades as an important broad-spectrum marker for immunocytochemical identification of normal and neoplastic NE cells. Synaptophysin, which initially was found in small-vesicle membranes of neurons, has also been demonstrated in NE cells, although in a smaller amount than chromogranin A.3Jahn R Schiebler W Ouimet CH Greengard P A 38,000-dalton membrane protein (p 38) present in synaptic vesicles.Proc Natl Acad Sci USA. 1985; 82: 4137-4141Crossref PubMed Scopus (731) Google Scholar, 4Wiedenmann B Franke WW Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles.Cell. 1985; 41: 1017-1028Abstract Full Text PDF PubMed Scopus (1227) Google Scholar, 5Wiedenmann B Waldherr R Buhr H Hille A Rosa P Huttner WB Identification of gastroenteropancreatic neuroendocrine cells in normal and neoplastic human tissue with antibodies against synaptophysin, chromogranin A, secretogranin I (chromogranin B), and secretogranin II.Gastroenterology. 1988; 95: 1364-1372Abstract PubMed Scopus (70) Google Scholar, 6Buffa R Rindi G Sessa F Gini A Capella C Jahn R Navone F De Camilli P Solcia E Synaptophysin immunoreactivity and small clear vesicles in neuroendocrine cells and related tumours.Mol Cell Probes. 1988; 2: 367-381Google Scholar, 7Portela-Gomes GM Stridsberg M Johansson H Grimelius L Co-localization of synaptophysin with different neuroendocrine hormones in the human gastrointestinal tract.Histochem Cell Biol. 1999; 111: 49-54Crossref PubMed Scopus (24) Google Scholar Synaptic vesicle protein 2 (SV2), like synaptophysin, is an integral membrane glycoprotein. It was initially identified in the central and peripheral nervous systems of different animal species from fish to mammals, as well as in the rat pancreas, anterior pituitary lobe, and adrenal medulla, and in some murine NE cell lines.8Buckley K Kelly RB Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells.J Cell Biol. 1985; 100: 1284-1294Crossref PubMed Scopus (575) Google Scholar, 9Lowe W Madeddu L Kelly RB Endocrine secretory granules and neuronal synaptic vesicles have three integral membrane proteins in common.J Cell Biol. 1988; 106: 51-59Crossref PubMed Scopus (147) Google Scholar, 10Hou XE Dahlström A Synaptic vesicle proteins in cells of the sympathoadrenal lineage.J Auton Nerv Syst. 1996; 61: 301-312Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar This glycoprotein occurs in three well-characterized isoforms, SV2A, SV2B, and SV2C. SV2A is widely distributed in the nervous system, in virtually all neurons. SV2B is also widely expressed in SV2A-containing neurons, although not as widely as SV2A, whereas SV2C is only observed in a small number of neurons in a few brain areas.11Bajjalieh SM Peterson K Linial M Scheller RH Brain contains two forms of synaptic vesicle protein 2.Proc Natl Acad Sci USA. 1993; 90: 2150-2154Crossref PubMed Scopus (156) Google Scholar, 12Bajjalieh SM Frantz GD Weimann JM McConnel SK Scheller RH Differential expression of synaptic vesicle protein 2 (SV2) isoforms.J Neurosci. 1994; 14: 5223-5235Crossref PubMed Google Scholar, 13Janz R Hofmann K Südhof C SVOP, an evolutionarily conserved synaptic vesicle protein, suggests novel transport functions of synaptic vesicles.J Neurosci. 1998; 18: 9269-9281Crossref PubMed Google Scholar, 14Janz R Südhof C SV2C is a synaptic vesicle protein with an unusually restricted localization: anatomy of a synaptic vesicle protein family.Neuroscience. 1999; 94: 1279-1290Crossref PubMed Scopus (172) Google Scholar Ultrastructurally, both chromogranin A and synaptophysin have been demonstrated in the secretory granules of endocrine cells.15Varndell IM Lloyd RV Wilson BS Polak JM Ultrastructural localization of chromogranin: a potential marker for the electron microscopic recognition of endocrine cell secretory granules.Histochem J. 1985; 17: 981-992Crossref PubMed Scopus (108) Google Scholar, 16Kalina M Lukinius A Grimelius L Höög A Falkmer S Ultrastructural localization of synaptophysin to the secretory granules of normal glucagon and insulin cells in human islets of Langerhans.Ultrastruct Pathol. 1991; 15: 215-219Crossref PubMed Scopus (18) Google Scholar SV2 has been observed ultrastructurally in vesicular structures in the mammalian nervous system8Buckley K Kelly RB Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells.J Cell Biol. 1985; 100: 1284-1294Crossref PubMed Scopus (575) Google Scholar and in the membranes of the secretory granules in an NE cell line from rat pheochromocytoma.9Lowe W Madeddu L Kelly RB Endocrine secretory granules and neuronal synaptic vesicles have three integral membrane proteins in common.J Cell Biol. 1988; 106: 51-59Crossref PubMed Scopus (147) Google Scholar, 17Tanner VA Plough T Tao-Cheng JH Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM immunocytochemistry for cell cultures.J Histochem Cytochem. 1996; 12: 1481-1488Crossref Scopus (60) Google Scholar, 18Marxen M Maienschein V Volknandt W Zimmermann H Immunocytochemical localization of synaptical proteins at vesicular organelles in PC12 cells.Neurochem Res. 1997; 22: 941-950Crossref PubMed Scopus (15) Google Scholar In man, SV2 has been found in the nervous system, but there are no reports about its occurrence in the NE cell system. The present study was therefore undertaken to ascertain the existence of SV2 in the human NE cell system, and to evaluate the extent to which it can be used as a broad-spectrum marker in normal and neoplastic NE cells. Tissue specimens from adult human gastric corpus and antrum, proximal duodenum, distal ileum, sigmoid colon, and pancreas were obtained from surgical samples removed at operations for adenocarcinoma. The specimens examined were taken from macroscopically normal tissue at least 2 to 5 cm from the neoplasm. Further tissue specimens from the pituitary, thyroid, parathyroid, and adrenal glands were also included in the study. The tissue specimens from the various organs were collected from three to six different cases. The pituitary glands were taken from autopsy cases without endocrine disturbances. The thyroid and adrenal tissues were from patients suffering from nonfunctional follicular and cortical adenoma, respectively. The parathyroid tissues were biopsy samples from histologically normal glands associated with a parathyroid adenoma. Hematoxylin and eosin (H&E)-stained sections from each organ showed normal histology. Forty-four human tumor specimens were also analyzed regarding their content of SV2-immunoreactive cells. The following tumor types were studied: various carcinoid tumors from the gastrointestinal and respiratory tracts, islet cell tumors, medullary thyroid carcinomas, anterior pituitary tumors, and pheochromocytomas. The ECLomas included were associated with enterochromaffin-like cell and gastrin cell hyperplasia, and the carcinoids of the ileum, proximal colon, and appendix were of the midgut (classical) type. The hindgut carcinoids showed a predominantly ribbon pattern. The bronchial carcinoids were centrally located. All carcinoid tumors displayed synaptophysin immunoreactivity, and all of them, except hindgut carcinoids, also showed chromogranin A immunoreactivity (see Results). Further, three cases of nesidioblastosis, in children with persistent neonatal hyperinsulinemic hypoglycemia, were included in the study; two were of the focal type and one was diffuse. All specimens were routinely fixed in 10% buffered neutral formalin, and some pancreatic specimens also in Stefanini's fixative (neutral picric acid-formaldehyde).19Stefanini M De Martino C Zamboni L Fixation of ejaculated spermatozoa for electron microscopy.Nature. 1967; 216: 173-174Crossref PubMed Scopus (1226) Google Scholar Some tissue specimens from the antrum, duodenum, and ileum were also fixed in Bouin's fluid. In addition, two pancreatic specimens were also fixed in buffered 2% glutaraldehyde, or in a mixture of 0.5% glutaraldehyde/4% formaldehyde. The fixation time was 18 to 20 hours at room temperature, followed by dehydration and embedding in paraffin. Sections 5-μm thick were cut and attached to poly-l-lysine-coated or to positively charged (Superfrost+; Menzel Gläser, Braunschweig, Germany) glass slides. The tissue sections were stained with H&E or immunostained by different methods to demonstrate various NE secretory granule products. The streptavidin-biotin-peroxidase complex technique,20Hsu SM Raine T Fanger H Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled (PAP) procedures.J Histochem Cytochem. 1981; 29: 577-580Crossref PubMed Scopus (13469) Google Scholar with diaminobenzidine as chromogen, was applied as a single immunostain mainly to reveal the distribution pattern of positive endocrine cell types in the respective regions, as well as to perform the control stainings specified below. For SV2 and synaptophysin immunostaining, the formalin- and Stefanini-fixed sections were pretreated in a microwave oven (Philips Whirlpool Nordic AB, Stockholm, Sweden) for 2 × 5 minutes at 750 W, using a Tris buffer, pH 8.0, as retrieval solution; this processing step was necessary to get satisfactory immunostaining of SV2 and synaptophysin. In the Bouin's-fixed sections the SV2 immunoreactivity seemed strong without microwave treatment. In co-localization studies, double-immunofluorescence methods were used without microwave pretreatment. The immunofluorescence staining of SV2 was enhanced by the catalyzed reporter deposition (CARD) method with biotinyl tyramide21Bobrow MN Harris TD Shaughnessy KJ Litt GJ Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays.J Immunol Methods. 1989; 125: 279-285Crossref PubMed Scopus (663) Google Scholar, 22Adams JC Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains.J Histochem Cytochem. 1992; 40: 1457-1463Crossref PubMed Scopus (572) Google Scholar as described below. For double-immunofluorescence staining, the sections were incubated with a cocktail of two antibodies: SV2 (anti-mouse) plus polyclonal antibody overnight at room temperature → biotinylated goat anti-mouse IgG, 30 minutes at room temperature → streptavidin-horseradish peroxidase, 30 minutes at room temperature → biotinyl tyramide, 10 minutes at room temperature → a mixture of streptavidin-Texas Red plus fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. Before applying the respective primary antibodies, the sections were incubated with nonimmune sera from the animal species producing the secondary antibodies at a dilution of 1:10. The secondary antibody in question was pre-incubated overnight at 4°C with 10 μl/ml normal serum both from the animal species recognized by the other secondary antibody and from the species producing the other secondary antibody. When two primary monoclonal antibodies raised in the same species (mouse) had to be used, a double-CARD method was used as follows: primary anti-mouse SV2 antibodies were applied overnight at room temperature, followed by biotinylated goat anti-mouse IgG and the CARD method with biotinyl tyramide and streptavidin-Texas Red as described above. Thereafter, the sections were incubated first with unlabeled avidin (100 μg/ml) overnight at room temperature, and then with the second primary anti-mouse antibody, followed by biotinylated horse anti-mouse IgG and the CARD enhancement method with biotinyl tyramide and streptavidin-FITC as chromogen. The avidin at a concentration of 100 μg/ml applied overnight was found to saturate the first-step biotin. Between each of the staining steps the sections were carefully washed with phosphate-buffered saline. The control stainings included: 1) omission of one or both of the primary antisera, 2) replacement of the first layer of antibody by nonimmune serum diluted 1:10 and by the diluent alone, and 3) preincubation (24 hours) of primary antiserum with the relevant antigen (10 nmol per ml diluted antibody solution) before application to the sections. The secondary antibodies were tested in relation to the specificity of the species in which the primary antibodies had been raised, the secondary antibody in question being replaced by secondary antibodies from different animal species. These control tests were performed with both immunofluorescence and streptavidin-biotin-peroxidase complex techniques. A neutralization test with SV2 antibodies was not performed, because we did not have access to SV2 antigen, but these antibodies have been characterized by Buckley and Kelly.8Buckley K Kelly RB Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells.J Cell Biol. 1985; 100: 1284-1294Crossref PubMed Scopus (575) Google Scholar Pancreatic tissue specimens, ∼1 mm3 in size, from two adult patients with no metabolic disease, were collected and fixed in 4% paraformaldehyde/0.5% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.2, supplemented with 0.1 mol/L sucrose, for 4 hours at 4°C. During subsequent dehydration in 50 to 95% ethanol, the temperature was lowered to −20°C. The specimens were infiltrated at −20°C with monomeres of the low-temperature hydrophilic-embedding medium Lowicryl K4 mol/L (Agar Scientific Ltd., Stansted, Essex, UK). Polymerization was performed in ultraviolet light (360 nm) at −20°C for 24 hours and at +20°C for another 48 hours.23Kellenberger E Carlemalm E Villiger W Roth J Garavito RM Low Denaturation Embedding for Electron Microscopy of Thin Sections. Chemishe Werke Lowi, Waldkreiburg1980Google Scholar, 24Lukinius A Wilander E Westermark G Engström U Westermark P Co-localization of islet amyloid polypeptide and insulin in the B cell secretory granules of the human pancreatic islets.Diabetologia. 1989; 32: 240-244Crossref PubMed Scopus (339) Google Scholar Islets in the adult pancreases were localized in semithin toluidine blue-stained sections. Ultrathin sections were cut with a diamond knife and placed on formvar-coated nickel grids. The immunogold labeling method used has been described in detail previously.25Lukinius A Ericsson JLE Grimelius L Korsgren O Ultrastructural studies of the ontogeny of fetal human and porcine endocrine pancreas, with special reference to colocalization of the four major islet hormones.Dev Biol. 1992; 153: 376-385Crossref PubMed Scopus (102) Google Scholar The sections were blocked for unspecific binding by applying nonimmune serum at a dilution of 1:10, followed by incubation overnight with the primary antibody diluted 1:50 in 0.05 mol/L Tris-buffered saline (TBS), pH 7.2, supplemented with 0.1 mol/L bovine serum albumin (BSA; Sigma, St. Louis, MO). After thorough rinsing in 0.05 mol/L TBS, pH 7.2, with 0.2% BSA, and in TBS, pH 8.2, with 1% BSA, the sections were incubated with 10- or 15-nm gold-conjugated goat anti-mouse IgG diluted 1:20 in TBS, pH 8.2, with 1% BSA, for 2 hours at 20°C. Again, the sections were thoroughly rinsed in TBS, pH 7.2, and finally were contrasted with 4% aqueous uranyl acetate and Reynolds lead citrate before examination in a Philips 201 electron microscope (Philips Industrial Electronics AB, Eindoven, The Netherlands). For controls, the primary antibody was omitted or was replaced with nonimmune serum. To improve the immunoreactivity, different dilutions of antibodies, durations of the incubation, and microwave pretreatments of the sections (at 150 to 750 W, for 30 seconds to 5 minutes) were tested. The primary antibodies are characterized in Table 1. The monoclonal antibodies to SV2 were a generous gift from Dr. R. B. Kelly (Department of Biochemistry and Biophysics, University of California, San Francisco, CA) and have been characterized by Buckley and Kelly.8Buckley K Kelly RB Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells.J Cell Biol. 1985; 100: 1284-1294Crossref PubMed Scopus (575) Google Scholar The other primary antibodies used have been characterized previously.7Portela-Gomes GM Stridsberg M Johansson H Grimelius L Co-localization of synaptophysin with different neuroendocrine hormones in the human gastrointestinal tract.Histochem Cell Biol. 1999; 111: 49-54Crossref PubMed Scopus (24) Google ScholarTable 1Antisera Used Antibody raised toCode no.Directed to amino acid sequenceWorking dilutionSourceSynthetic adrenocorticotropin†antisera raised in mouse (monoclonal),M3501 clone 02A31–391:10DAKO, Santa Barbara, CASynthetic human calcitonin‡antisera raised in rabbit;A05761–321:80DAKOSynthetic cholecystokinin‡antisera raised in rabbit;B 38-110–201:40Eurodiagnostica, Malmö, SwedenHuman chromogranin A†antisera raised in mouse (monoclonal),LK2H10-1:20Boehringer Mannheim, Mannheim, GermanyPurified human follicle-stimulating hormone†antisera raised in mouse (monoclonal),M3504 clone C10β-subunit1:5DAKOSynthetic porcine gastric inhibitory polypeptide‡antisera raised in rabbit;B35-1-1:80EurodiagnosticaSynthetic human gastrin I‡antisera raised in rabbit;B36-1C terminal1:80EurodiagnosticaSynthetic human gastrin I‡antisera raised in rabbit;61050N terminal (2–17)1:800Peninsula Laboratories Europe, Merseyside, UKSynthetic human gastrin I§antisera raised in guinea-pig.B-GP360-1C terminal1:240EurodiagnosticaSynthetic porcine glucagon‡antisera raised in rabbit;A565 lot 081-1:60DAKOSynthetic human glucagon/glicentin†antisera raised in mouse (monoclonal),Glu-001 7360061Midportion (5–15)1:20Novo Nordisk S/A, Bagsvaerd, DenmarkPurified human growth hormone§antisera raised in guinea-pig.4749-9509-1:200Biogenesis Ltd., Poole, UKSynthetic human insulin§antisera raised in guinea-pig.Ma 47A chain1:80P. Westermark, Dept. of Pathology, Uppsala, SwedenPurified human luteinizing hormone†antisera raised in mouse (monoclonal),5720-1004-'pre-diluted' 1:2BiogenesisSynthetic neurotensin‡antisera raised in rabbit;6-8208C terminal (8–13)1:50E. Theodorsson, Dept. of Clin. Chemistry, Linköping, SwedenSynthetic human pancreatic polypeptide‡antisera raised in rabbit;A619 lot 105-1:50DAKOSynthetic parathyroid hormone‡antisera raised in rabbit;41P1–341:5BioGenex, San Ramon, CASynthetic porcine PYY§antisera raised in guinea-pig.B-GP520-1-1:800EurodiagnosticaPurified human prolactin†antisera raised in mouse (monoclonal),MCA713 clone INN–hPRL-3-1:15Serotec, Oxford, UKPurified bovine S-100‡antisera raised in rabbit;Z-311, lot 113-1:80DAKOSynthetic porcine secretin‡antisera raised in rabbit;B-33-1-1:10EurodiagnosticaSynthetic serotonin*Antisera raised in rat (monoclonal).YC5/45-1:20Medicorp, Montreal, CanadaSynthetic human somatostatin‡antisera raised in rabbit;A 566, lot 721–141:100DAKOSynaptic vesicle protein 2 (SV2)†antisera raised in mouse (monoclonal),--1:10R.B. Kelly, Dept. of Biochemistry and Biophysics, UCSF, CABovine synaptophysin†antisera raised in mouse (monoclonal),SY38-1:20Boehringer MannheimPurified human thyroid stimulating hormone†antisera raised in mouse (monoclonal),8920-0584-'pre-diluted'BiogenesisPurified rat tyrosine hydroxylase†antisera raised in mouse (monoclonal),1017 381-1:5Boehringer MannheimDilutions used in immunofluorescence staining; for the ABC staining the dilutions were 10 to 20 times higher.* Antisera raised in rat (monoclonal).† antisera raised in mouse (monoclonal),‡ antisera raised in rabbit;§ antisera raised in guinea-pig. Open table in a new tab Dilutions used in immunofluorescence staining; for the ABC staining the dilutions were 10 to 20 times higher. The labeled secondary antisera were as follows: biotinylated swine anti-rabbit IgG, biotinylated goat anti-mouse IgG, streptavidin biotin complex kit (DAKO, Glostrup, Denmark), unlabeled avidin, biotinylated horse anti-mouse and Texas Red- and FITC-labeled streptavidin (Vector Laboratories, Burlingame, CA), FITC-conjugated goat anti-rabbit IgG (Sigma Chemical Co.), biotinyl tyramide (Dupont-New England Nuclear Research Products, Boston, MA), and 10- or 15-nm gold-conjugated goat anti-mouse IgG (GAM-G10 and GAM-G15; Amersham International, Amersham, Bucks, England). SV2-immunoreactive cells were demonstrated with both streptavidin-biotin-peroxidase complex and immunofluorescence methods with the fixatives used, except glutaraldehyde. The strongest immunoreactivity was seen in the Bouin's-fixed tissues. The staining intensity became gradually weaker with Stefanini's fixative and formalin, and was only faint with the glutaraldehyde/paraformaldehyde mixture. When either microwave pretreatment of the sections or the CARD technique was used, the staining intensity in the Stefanini- and formalin-fixed tissues increased to a level similar to that seen in Bouin's-fixed tissue. The enhancement of the staining intensity did not, however, influence the frequency of immunoreactive cells. Microwave pretreatment of pancreatic sections fixed in 2% glutaraldehyde or in a glutaraldehyde/paraformaldehyde mixture did not improve the immunostaining. Control Stainings: In double-immunofluorescence staining, omission of one of the primary antibodies gave a staining pattern corresponding to that obtained with the remaining primary antibody. The other staining controls were negative. When using double-CARD immunostaining, biotin blocking is necessary, to avoid unspecific binding of the secondary antibody of the second staining sequence, as demonstrated in Figure 1, Figure 2.Figure 2Human pancreatic islet immunostained with double-CARD for SV2 (A) and chromogranin A (B) with avidin blocking of biotin after the first staining sequence. The staining pattern in A differs from that in B. After the avidin blocking of biotin, there is a distinct difference in the staining pattern between A and B. Scale bar, 170 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Distribution of SV2-Immunoreactive Endocrine Cells of Various Organs: SV2-immunoreactive cells were observed in all organs examined, ie, in the gastrointestinal tract, pancreas, pituitary, thyroid, parathyroid glands, and adrenal medulla, but not in the adrenal cortex. SV2 immunoreactivity was diffusely distributed in the entire cytoplasm, involving processes of cells when present. In addition, SV2 immunostaining visualized nerve structures in all organs examined. The whole gastrointestinal tract contained scattered SV2-immunoreactive cells, localized at all levels of the mucosa, but these cells were most numerous in the middle third portion. The highest frequency was found in the antrum, followed by the duodenum and colon. Few SV2-immunoreactive cells were observed in Brunner's glands and in the gastric corpus and ileum. The results concerning the co-localization of SV2 with hormones in the gastrointestinal endocrine cells are summarized in Table 2. In the corpus, the serotonin (enterochromaffin) cells and occasionally somatostatin cells displayed SV2 immunoreactivity. In the antrum, most enterochromaffin cells, as well as virtually all somatostatin and gastrin cells (Figure 3), were also immunoreactive. In the duodenum, only some of the enterochromaffin cells but virtually all gastrin, cholecystokinin (CCK), secretin, and enteroglucagon cells were SV2-immunoreactive; occasionally gastric inhibitory polypeptide cells were immunoreactive, whereas somatostatin cells were negative (Figure 4). In Brunner's glands the gastrin and CCK cells were SV2-immunoreactive, as well as most enterochromaffin cells. The sparse SV2-immunoreactive cells seen in the ileum represented enterochromaffin or enteroglucagon cells, and a few of them neurotensin cells, but the somatostatin cells were nonimmunoreactive. In the colon, enteroglucagon and peptide tyrosine tyrosine cells and most enterochromaffin cells were SV2-positive, but not the somatostatin cells.Table 2Co-Localization of SV2 with Hormones in Various Endocrine Cell Types in Different Parts of the Human Gastrointestinal Tract and in the PancreasCell typeGastrointestinal levelSerotonin (EC)GastrinSomato- statinCCKGIPSecretinEGEG//PYYNeuro- tensinInsulin +Glucagon +PPCorpus++(+)Antrum++/+++++/+++++/+++Duodenum: villi+ crypts++++—+++(+)++++/+++ Brunner's glands+++++—+++Ileum: villi+ crypts++—+++Colon+/+++++Pancreas++/++++++EC, enterochromaffin cells; CCK, cholecystokinin; GIP, gastric inhibitory polypeptide; EG, enteroglucagon (glucagon/glicentin); PYY, peptide tyrosine tyrosine; PP, pancreatic polypeptide.The number of SV2-immunoreactive cells is graded in relation to the total number of corresponding endocrine cells at each gastrointestinal level: +++, more than 50% of the endocrine cells were SV2-immunoreactive (positive); ++, 30–50% of the cells were positive; (+), occasional cells were positive; −, no SV2-immunoreactive endocrine cells. Open table in a new tab Figure 4Human duodenal villi double-immunostained for SV2 (Texas Red) and somatostatin (FITC), showing that the somatostatin cells are SV2-nonreactive. Scale bar, 170 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) EC, enterochromaffin cells; CCK, cholecystokinin; GIP, gastric inhibitory polypeptide; EG, enteroglucagon (glucagon/glicentin); PYY, peptide tyrosine tyrosine; PP, pancreatic polypeptide. The number of SV2-immunoreactive cells is graded in relation to the total number of corresponding endocrine cells at each gastrointestinal level: +++, more than 50% of the endocrine cells were SV2-immunoreactive (positive); ++, 30–50% of the cells were positive; (+), occasional cells were positive; −, n
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