Immunocytochemical identification of nuclear structures containing snRNPs in isolated rat liver cells

Small nuclear ribonucleoproteins (snRNPs) containing U1, U2, U4, U5, and U6 small nuclear RNAs were detected by ultrastructural immunocytochemistry in the nuclei of isolated rat hepatocytes using Fab fragments of anti-Sm and anti-RNP autoantibodies. Their localization was carried out in normal cells and in cells treated with two drugs, the adenosine analog DRB and CdCl2, which alter the number and distribution of nuclear RNP components. It was found that more precise determination of the distribution of these small RNAs could be obtained by using two complementary procedures in parallel rather than either one alone. They consisted of an indirect immunoperoxidase labeling carried out before embedment and an indirect immunogold labeling applied to thin sections of cells embedded in Lowicryl K4M. The results indicate that snRNPs are associated with all extranucleolar perichromatin fibrils and granules and interchromatin fibrils, which confirms that they occur in structures involved in the synthesis and processing of hnRNA. The snRNPs are not associated with nucleolar perichromatin granules induced by DRB, which confirms that there may be two kinds of perichromatin granules. The snRNPs are also associated with the still enigmatic interchromatin granules which apparently do not contain hnRNA but at least in DRB-treated cells, also contain ribosomal RNA.     

Chromogranin in bronchopulmonary neuroendocrine cells. Immunocytochemical detection in human, monkey, and pig respiratory mucosa

Immunoreactive chromogranin A was demonstrated by immunocytochemistry in the cytoplasm of neuroendocrine cells (NEC) and neuroepithelial bodies (NEB) in human, monkey, and pig respiratory mucosa. Three different antisera (one monoclonal and one polyclonal to human chromogranin A, and one polyclonal to bovine chromogranin A) were applied in this study. Chromogranin immunopositivity varied in extent and intensity according to the antiserum applied and the tissue investigated. The monoclonal antibody revealed the strongest immunoreaction. Good correlation between chromogranin immunoreactivity and Grimelius silver staining was observed by comparing adjacent sections, although more cells seemed to reveal chromogranin immunoreactivity than argyrophylia. Chromogranin appears to be a useful histological marker for APUD cells in the respiratory mucosa of several species.     

Immunocytochemical localisation of pancreastatin and chromogranin A in porcine neuroendocrine tissues.

Pancreastatin is a 49 amino acid peptide with a C-terminal glycine amide originally isolated from porcine pancreas. In the present study the cellular localisation of pancreastatin in porcine neuroendocrine tissue was examined immunocytochemically using an antiserum raised against porcine pancreastatin (33-49) that does not cross-react with porcine chromogranin A. In order to study the possible precursor-product relationship between chromogranin A and pancreastatin the cellular localisation of both peptides was examined in peripheral tissues using simultaneous double immunostaining. The pancreastatin antiserum immunostained cells and nerve fibers throughout the neuroendocrine system. In most of the examined tissues we found colocalisation of pancreastatin and chromogranin A immunostaining. These results support the precursor-product concept for chromogranin A and pancreastatin. However, in the gastrointestinal tract and the adenohypophysis a minor population of the endocrine cells exhibited immunostaining with only one of the two antibodies. This discrepancy between immunostaining with pancreastatin antiserum and monoclonal chromogranin A antibody could be due to absence of, or extensive, processing of chromogranin A in certain cell populations.     

Immunocytochemical identification of murine and human megakaryocyte colonies in soft-agar cultures

Summary Murine megakaryocyte (MK) colonies in soft-agar cultures were immunocytochemically stained with platelet antiserum and an immuno-alkaline phosphatase procedure. Subsequently, cytochemical staining for acetylcholinesterase was used to confirm the specificity of the immunolabelling technique. The correlation of numbers of megakaryocyte colonies enumerated by independent observers was excellent. A comparable platelet antiserum directed against human platelet epitopes was utilized to identify human MK colonies in soft-agar cultures of human bone marrow. Using this method, we determined that the frequency of detectable human MK colonies in our agar culture system was maximal between days 10 and 12. The immunocytochemical staining technique we have developed for identification of MK colonies in soft-agar cultures yielded good cellular morphology and produced an intensely specific label against a clear background; it therefore facilitated accurate enumeration of MK colonies. This non-fluorescent method avoids dependence upon a non-permanent marker, and allows the simultaneous enumeration of positive and negative colonies.     

Immunocytochemical and biochemical identification of angiotensin II in human nasal tissue

Angiotensin II (ANG II) was identified immunocytochemically and biochemically in biopsy samples of human nasal tissue. Staining for ANG II was predominantly found in structures similar to a string of pearls with consecutive short varicose areas, which is characteristic for neuronal tissue. The localization of ANG II in neurons was confirmed by positive staining of adjacent tissue sections with a specific antibody to neurofilament or doublestaining with both antibodies in one section. Likewise, ANG II-like material was also determined radioimmunologically in nasal tissue extracts. The concentrations of ANG II varied form 1.28 to 332.78 fmol/g wet tissue weight with an average concentration of 79.61+/-44.09 fmol ANG II/g wet tissue weight (mean+/-SEM, n=7). The ANG II-immunoreactive material was further characterized biochemically by HPLC on a reversed phase C(18) column in an acetonitrile and methanol gradient as Ile(5)-ANG II and ANG II metabolites such as Ile(4)-ANG III, Ile(3)-ANG II(3-8)hexapeptide and Ile(2)-ANG II(4-8)pentapeptide.     

Immunocytochemical identification of cell types in human mammary gland: variations in cellular markers are dependent on glandular topography and differentiation

Antiserum to epithelial membrane antigen and three monoclonal antibodies (MAb) to milk-fat globule membranes immunocytochemically stain only epithelial cells, whereas a fourth reacts also with myoepithelial cells in inter- and intralobular ducts of human breast. Staining with peanut lectin shows a gradual increase for epithelial cells, from little or no staining in ducts through variable staining in ductules to intense staining in secretory alveoli. Antisera and MAb to vimentin, smooth-muscle actin, MAb to the common acute lymphoblastic leukemia antigen and to a glycoprotein of 135 KD stain myoepithelial cells in main ducts, but this staining is reduced in inter- and intralobular ducts and ductules. MAb to epithelial-specific keratin 18 stain a minor population of ductal epithelial cells, the major population of epithelial cells in interlobular (ILD) and extralobular terminal ducts (ETD), and epithelial cells in a minority of ductules. In lactating glands most epithelial cells in ductules are stained, but the alveolar and myoepithelial cells are unstained. Keratin MAb PKK2 and LP34 strongly stain myoepithelial cells, but only a minor population of epithelial cells in main ducts. However, these MAb stain principally the epithelial cells in ILD, ETD, and a minority of ductules. In lactating glands most epithelial cells are stained in ductules, but the myoepithelial and not the alveolar cells are stained intensely in secretory lobules. It is suggested that the unusual staining pattern of cells found principally in the ILD, ETD, and some ductules may represent regions of growth and/or subpopulation(s) of cells intermediate between epithelial and myoepithelial cells.     

Immunocytochemical identification of laticifers

Summary sensitive immunocytochemical method for the identification of laticifers has been developed. Frozen sections of various laticifer-bearing plant material, mounted on slides, were first flooded with the IgG fraction of rabbit anti-latex antiserum, prepared using whole latex ofAsclepias syriaca, then flooded with fluorescein-conjugated IgG fraction goat anti-rabbit IgG to visualize laticifers. Positive fluorescence was observed for laticifers in shoots and embryos ofA. syriaca andStapelia bella and embryos ofA. tuberosa. Laticifers did not fluoresce in shoots ofA. tuberosa andEuphorbia tirucalli, in embryos ofE. marginata, or in petioles ofMusa paradisiaca andCichorium intybus. Controls prepared with uninjected rabbit serum were negative (no fluorescence).     

Immunocytochemical prognostic markers in pituitary tumors

The value of different immunocytochemical markers in the evaluation of the pituitary tumor aggressiveness is discussed on the basis of the data from the literature and the author's personal experience. The usefulness of the proliferation markers (PCNA, Ki-67), prothymosin-alpha, the endothelial markers revealing the intratumoral vascularization and of the expression or co-expression of glycoprotein hormones in predicting the aggressive behaviour of the tumor is likely, but still needs further studies.     

Immunocytochemical identification of structures containing putative red pigment-concentrating hormone in two species of decapod crustaceans

Summary By use of an antiserum raised against the Nterminal sequence pGlu-Leu-Asn-Phe..., common to red pigment-concentrating hormone (RPCH) of Pandalus borealis and three structurally similar insect neuropeptides, putative RPCH-immunopositive structures were revealed in the eyestalks of Carcinus maenas and Orconectes limosus and in the brain and thoracic ganglion (TG) of C. maenas. In the eyestalks, complete neurosecretory pathways were demonstrated, consisting of perikarya, axons and terminals in the neurohemal organ, the sinus gland (SG). In C. maenas approximately 20 small RPCH cells are present as a distinct group adjacent to the medulla terminalis ganglionic X-organ (MTGXO, XO). They are morphologically different from the larger XO perikarya, which contain the crustacean hyperglycemic hormone (CHH). The occurrence of both neuropeptides in distinct neurosecretory pathways was ascertained by immunologic double staining (PAP/gold) or by analysis of consecutive sections. In addition, a group of two to four larger RPCH cells is located in the proximal part of the MT. In O. limosus, RPCH cells are found in the XO. Cells corresponding to the proximal MT cells of C. maenas were not found. In both species, a few more weakly staining immunopositive perikarya were observed in clusters of cell somata of the optic ganglia. It is uncertain whether these are connected to the SG.In the brain of C. maenas, several smaller and three larger perikarya were consistently observed in the dorsal lateral cell somata adjacent to the olfactory lobes. In the optic nerve, two axons that project into the eyestalk were stained. Some axons were also observed in the ventral median neuropil of the brain. In the TG, RPCH cells were found in small numbers in median positions, i.e., in clusters of somata between the ganglia of the appendages.HPLC analysis of the red pigment-concentrating activity from the SG of C. maenas revealed that the retention time of the neuropeptide is similar but not identical to that of Pandalus borealis RPCH.     

Immunocytochemical identification of abnormal polymorphonuclear neutrophils in patients with leukemia.

High titer, monospecific antibodies to human granulocyte myeloperoxidase, cathepsin G, elastase, lysozyme, and lactoferrin were conjugated with fluorescein and rhodamine and used for immunofluorescent staining of mature neutrophils obtained from 25 patients with acute and chronic leukemia. In 11 (44%) of the patients, two populations of mature neutrophils were detected. The abnormal cells were identified by complete deficiency of one or more markers and constituted 10%-100% of the total number of neutrophils. This immunocytochemical approach may permit recognition of mature cells derived from leukemic clones, and serial determinations of the ratio of normal to abnormal cells may be useful in the management of patients with leukemia.     

Immunocytochemical identification of polypeptide YY (PYY) cells in the human gastrointestinal tract

Various parts of the human gastrointestinal tract were investigated immunocytochemically for the occurrence of polypeptide YY (PYY) and pancreatic polypeptide (PP). PYY-immunoreactive cells were observed in the lower part of the ileum, in the colon and in the rectum, and PP-immunoreactive cells were found in the colon and rectum. Both cell types were of the open type, i.e. they extended from the basal lamina to the gut lumen. PYY-immunoreactive cells were seen to emit cytoplasmic processes to the neighbouring goblet cells. This latter observation suggests that PYY cells may exert a paracrine action on the mucus-secreting goblet cells. Staining of consecutive thin plastic sections and staining of the same section simultaneously for two peptides showed that PYY-immunoreactivity did not occur in PP- or enteroglucagon-immunoreactive cells. On the ultrastructural level PYY-immunoreactivity was localized in basal granulated endocrine cells. These cells contained round or slightly oval electron dense granules with a mean diameter of 150 nm (range 100-300 nm).     

Immunocytochemical localization of interferons in human trophoblast populations.

It is known that Interferon (IFN) is present in normal body fluids and tissues during pregnancy. Using an immunohistochemical technique and a panel of monoclonal antibodies we have localized IFN-alpha, -beta and -gamma directly on formalin-fixed paraffin-embedded normal human placentae at different stages of pregnancy and in the hydatidiform mole. The results show that IFNs is mostly localized in villous syncytiotrophoblast and in extravillous interstitial-trophoblast. No reactivity was observed in villous cytotrophoblast or in cytotrophoblast cell columns. The most intense staining was observed for IFN-alpha and -beta, while IFN-gamma was rather weak. There is then a gradual diminution in IFN reactivity with increasing gestation age being almost imperceptible at term. These results suggest that IFN may deploy antiviral, immunomodulator and differentiation activities during normal human pregnancy.     

Immunocytochemical localisation of transferrin in the human brain.

Immunocytochemical studies on the adult human brain have shown that transferrin is localized within three main compartments in the adult human brain. Oligodendrocytes and some astrocytes together with cells of the choroid plexus showed the highest intensity of staining. Neuronal staining occurred mainly within pyramidal or large polygonal cells, but this showed considerable regional variation being most marked in areas such as the cerebral cortex, amygdala, hippocampus, brainstem and cerebellar Purkinje cells. Small neurones such as caudate interneurones and granule cells showed relatively low activity. Diffuse immunostaining of the neuropil was evident, particularly where heavy neuronal or glial staining occurred. Immunostaining was also observed in white-matter fibre tracts. This pattern of distribution helps to provide a model for the mechanisms responsible for iron homeostasis in the normal brain.     

Immunocytochemical studies of spectrin in hamster cardiac tissue

The spectrins are a family of cytoskeletal-membrane proteins that have a wide tissue distribution. In the present study, we employed polyclonal antibodies made against mammalian and avian erythroid spectrins as well as mammalian brain spectrin to assess their presence and distributions in the mammalian heart. Western blot analyses revealed that all three antibodies were specific for a 240,000 molecular weight alpha-spectrin subunit found in hamster erythrocyte ghost homogenates, whole hamster heart, and isolated hamster cardiac myofibril homogenates. Spectrin staining was absent from the Triton X-100-extracted supernatant fraction of myofibril preparations, suggesting that the protein is linked to the myofibril precipitate after exposure to the detergent. Frozen, unfixed, 2-microns-thick; sections of adult. Syrian golden hamster cardiac tissue exhibited strong immunofluorescent staining of intercalated discs and Z-bands using all three antibodies. In addition, the mammalian erythroid spectrin antibodies showed staining of the sarcolemma, and in cross section, revealed a delicate internal network of staining that appears to surround individual myofibrils. This may be T-tubule-associated staining. Myofibrils isolated from cardiac myocytes using Triton X-100 show positive Z-band staining using all three antibodies. Double staining with Texas Red-labeled monoclonal desmin and FITC-labeled polyclonal spectrin antibodies revealed that both stained the myofibrillar Z-line regions. These results demonstrate that spectrin is closely associated with the membranes, myofibrils, and intermediate filaments in the mammalian heart.     

Immunocytochemical identification of amyloid in formalin-fixed paraffin sections

Summary Formalin-fixed paraffin sections of livers, spleens and kidneys from patients with primary, secondary and familial amyloidosis as well as from a casein-induced murine amyloid model were analysed by an immunocy-tochemical (unlabeled antibody enzyme) method utilizing antisera to amyloid-related proteins. All amyloid deposits of all amyloid types showed positive reactions with anti-AP of the respective species. Positive reaction of anti-human AA to human secondary amyloid deposits and of anti-mouse AA to the deposits of casein-induced murine amyloid was also observed, but there was no species cross reactivity. No significant deposition of the reaction products was produced by anti-immunoglobulin light chains on deposits of any amyloid type, or by anti-AA in the tissues from primary or familial amyloidosis. The results indicate that amyloid proteins AA and AP can survive as antigens through routine histologic preparation, that anti-AP can be a universal marker for deposits of any amyloid type within the same species, and that AA-type amyloid can be identified by this method while there may as yet be no feasible universal marker for the AL-type at present.Presented in part at the 64th Annual Meeting of the Federation of American Societies for Experimental Biology, Anaheim, California, April, 1980     

Immunocytochemical identification of dynorphin-containing vesicles in Brattleboro rats

Vasopressin and its carrier protein, vasopressin-associated neurophysin, are co-packaged together with an opioid peptide, dynorphin, into 160 nm diameter neurosecretory vesicles in the normal rat hypothalamo-neurohypophysial system. The homozygous Brattleboro rat lacks vasopressin and vasopressin-associated neurophysin, but contains substantial amounts of dynorphin in the vasopressin-deficient neurosecretory cells. We used post-embedding electron microscopic immunocytochemistry to determine the subcellular location of dynorphin in Brattleboro rats. The results show that dynorphin is present within 100 nm neurosecretory vesicles in homozygous Brattleboro cell bodies and axons, and within 160 nm vesicles in heterozygous (control) neurosecretory cell bodies and axons. Oxytocin-associated neurophysin is present in a separate population of magnocellular neurons in both homozygous and heterozygous rats, and is contained within 160 nm vesicles in both cases. Therefore, the absence of synthesis of the vasopressin prohormone results in a dramatic reduction of neurosecretory vesicle size, despite the continued synthesis and packaging of dynorphin peptides.     

Immunocytochemical identification of actin in rabbit spermiogenesis and spermatozoa

Actin was localized in testicular spermatids and in spermatozoa of rabbit by using a monoclonal anti-actin antibody and a specific antiserum against actin, labeled with colloidal gold. The antibody reactivity with sperm homogenates was determined by immunoblotting of one-dimensional gels. With on-grid postembedding immunostaining of Lowicryl K4M sections, actin was identified in the subacrosomal region of differentiating spermatids, and in four bulges situated between the inner acrosomal membrane and the nuclear envelope and in the anterior part of the postacrosomal region of ejaculated spermatozoa. Sperm actin was identified on two-dimensional gels as two spots in the isoelectric point and molecular weight corresponding to gamma and beta-isoforms of actin. Immunoblots stained with specific antibodies demonstrated that rabbit spermatozoa express gamma and beta-actin isoforms.     

Evidence of nanobacterial-like structures in calcified human arteries and cardiac valves

Mechanisms mediating vascular calcification remain incompletely understood. Nanometer scale objects hypothesized to be a type of bacteria (nanobacteria) are associated with calcified geological specimens, human kidney stones, and psammona bodies in ovarian cancer. Experiments were designed to evaluate human vascular tissue for the presence of similar nanometer-scale objects. Calcified human aneurysms (n = 8), carotid plaques (n = 2), femoral arterial plaques (n = 2), and cardiac valves (n = 2) and noncalcified aneurysms from patients with bicuspid aortic valve disease (n = 2) were collected as surgical waste from the Heart Hospital of Austin, Austin, Texas, and Mayo Clinic, Rochester, Minnesota. Whole mounts or adjacent sections from each specimen were examined by electron microscopy, stained for calcium phosphate, or stained with a commercially available antibody (8D10). Filtered (0.2 microm) homogenates of aneurysms were cultured and costained with 8D10 antibody followed by PicoGreen to detect DNA or incubated with [3H]uridine. Staining for calcium phosphate was heterogeneously distributed within all calcified tissues. Immunological staining with 8D10 was also heterogeneously distributed in areas with and without calcium phosphate. Analysis of areas with positive immunostaining identified spheres ranging in size from 30 to 100 nm with a spectral pattern of calcium and phosphorus (high-energy dispersive spectroscopy). Nanosized particles cultured from calcified but not from noncalcified aneurysms were recognized by a DNA-specific dye and incorporated radiolabeled uridine, and, after decalcification, they appeared via electron microscopy to contain cell walls. Therefore, nanometer-scale particles similar to those described as nanobacteria isolated from geological specimens and human kidney stones can be visualized in and cultured from calcified human cardiovascular tissue.     

Immunocytochemical evidence for glucagon-containing cells in the human stomach.

To determine if glucagon-containing cells could be identified in the human fundus, stomachs attained at autopsy within 4-hours of death from persons previously considered to be in good health were examined by the indirect immunoperoxidase technique using antiglucagon serum 30K. Glucagon-containing cells were demonstrated in one of eight gastric fundi examined. The glucagon content of acid alcohol extracts of the fundi examined. The glucagon content of acid alcohol extracts of the funci was low in all cases. Glucagon content was also low in canine stomach removed 4-hours after death. It is concluded that glucagon-containing cells, demonstrable by immunocytochemical techniques, may be present in the gastric fundus of humans.     

Isolation and identification of human brain natriuretic peptides in cardiac atrium

By using a radioimmunoassay (RIA) system newly established for human brain natriuretic peptide (BNP), a high concentration of immunoreactive (ir-) human BNP (hBNP) has been found in cardiac atrium (1). Two molecular forms of ir-hBNP of 4K and 13-15K were isolated from atrial extracts by using anti-hBNP IgG immunoaffinity chromatography and reverse phase high performance liquid chromatography (HPLC). By microsequencing, the peptides were determined to be a pro-hBNP (gamma-hBNP) and its C-terminal 32-amino acid peptide (hBNP-32). Based on these results, in cardiac atrium, hBNP is found to be processed in a pathway similar to that of porcine BNP (pBNP) but distinct from that of rat BNP, although low MW hBNP-32 is a major form in contrast to pBNP which exists as a high MW gamma-pBNP.