Methotrexate loading of red cell carriers by osmotic stress and electric-pulse methods: ultrastructural observations

Transmission and scanning electron microscopy (EM) of osmotically stressed and electrically pulsed human erythrocyte carriers indicated that the mode of uptake of methotrexate (MTX) varied depending on the method of carrier production. The preswell and electroporation loading methods, apparently by facilitating passive diffusion across the cell membrane, promoted incorporation of the MTX directly into the cytoplasm of the carriers, as evidenced by their variable electron densities. The preswell method produced carriers displaying leptocytic characteristics, whereas the electroporation method produced carriers exhibiting sphero- and stomatocytic transformation. Hypotonic dialysis-prepared carriers took up MTX primarily by endocytosis and secondarily by passive diffusion. Endocytotic activity was not induced by the MTX. Scanning EM revealed that most of the dialysis-prepared carriers exhibited prominent invaginations of the cell surface. Transmission EM of serial thin sections through these carriers revealed numerous fully internalized, membrane-bound vesicles. Endocytotic activity caused progressive loss of membrane and resulted in the sphero- and stomatocytic transformation of the carriers.     

Measurement of bioreactor perfusion using dynamic contrast agent-enhanced magnetic resonance imaging.

Dynamic magnetic resonance imaging was used to monitor solute diffusion through aggregates of Chinese hamster ovary cells growing on macroporous carriers in a fixed-bed bioreactor. Diffusion-weighted (1)H magnetic resonance imaging (MRI) and scanning electron microscopy demonstrated that cell growth in the bioreactor was heterogeneous, with the highest cell densities being found at the periphery of the carriers. T(1)-weighted magnetic resonance imaging measurements of the inflow of a commonly used magnetic resonance contrast agent, gadolinium-diethylenetriaminopentaacetic acid (Gd-DTPA), showed that migration of the agent through the peripheral cell masses could be explained by diffusion. However, appearance of the contrast agent in the center of the carriers was too fast to be explained by simple diffusion and indicated that these regions were perfused by convective flow. The average diffusivity of Gd-DTPA through the cell mass was found to be (2.4 +/- 0.2) x 10(-10) m(2) sec(-) (mean +/- SEM). This technique will be useful in the characterization and development of high-cell-density bioreactor systems, in which solute transport plays a critical role in cell growth and physiology.     

Imaging protein-protein interactions in living cells

The complex organization of plant cells makes it likely that the molecular behaviour of proteins in the test tube and the cell is different. For this reason, it is essential though a challenge to study proteins in their natural environment. Several innovative microspectroscopic approaches provide such possibilities, combining the high spatial resolution of microscopy with spectroscopic techniques to obtain information about the dynamical behaviour of molecules. Methods to visualize interaction can be based on FRET (fluorescence detected resonance energy transfer), for example in fluorescence lifetime imaging microscopy (FLIM). Another method is based on fluorescence correlation spectroscopy (FCS) by which the diffusion rate of single molecules can be determined, giving insight into whether a protein is part of a larger complex or not. Here, both FRET- and FCS-based approaches to study protein-protein interactions in vivo are reviewed.     

Multimodal analysis of Plasmodium knowlesi‐infected erythrocytes reveals large invaginations,swelling of the host cell,and rheological defects

The simian parasite Plasmodium knowlesi causes severe and fatal malaria infections in humans, but the process of host cell remodelling that underpins the pathology of this zoonotic parasite is only poorly understood. We have used serial block‐face scanning electron microscopy to explore the topography of P. knowlesi‐infected red blood cells (RBCs) at different stages of asexual development. The parasite elaborates large flattened cisternae (Sinton Mulligan's clefts) and tubular vesicles in the host cell cytoplasm, as well as parasitophorous vacuole membrane bulges and blebs, and caveolar structures at the RBC membrane. Large invaginations of host RBC cytoplasm are formed early in development, both from classical cytostomal structures and from larger stabilised pores. Although degradation of haemoglobin is observed in multiple disconnected digestive vacuoles, the persistence of large invaginations during development suggests inefficient consumption of the host cell cytoplasm. The parasite eventually occupies ~40% of the host RBC volume, inducing a 20% increase in volume of the host RBC and an 11% decrease in the surface area to volume ratio, which collectively decreases the ability of the P. knowlesi‐infected RBCs to enter small capillaries of a human erythrocyte microchannel analyser. Ektacytometry reveals a markedly decreased deformability, whereas correlative light microscopy/scanning electron microscopy and python‐based skeleton analysis (Skan) reveal modifications to the surface of infected RBCs that underpin these physical changes. We show that P. knowlesi‐infected RBCs are refractory to treatment with sorbitol lysis but are hypersensitive to hypotonic lysis. The observed physical changes in the host RBCs may underpin the pathology observed in patients infected with P. knowlesi.     

Role of vitamin E in glutathione-induced oxidant stress: methemoglobin, lipid peroxidation, and hemolysis.

Red blood cells (RBC) from normal and vitamin E-deficient rats were incubated in a hypertonic solution of reduced glutathione adjusted to pH 8. Methemoglobin formation occurred in intact RBC from both normal and vitamin E-deficient rats. Hemolysis was significantly greater in RBC from vitamin E-deficient rats. Experiments with catalase, superoxide dismutase, and methional showed that H(2)O(2) was the primary extracellular source of oxidant stress. Extracellular superoxide and hydroxyl radical were not involved in oxidant stress. Experiments with dimethyl sulfoxide showed that intracellular hydroxyl radical, generated from H(2)O(2), was the hemolytic agent. Neither methemoglobin formation nor lipid peroxidation involved hydroxyl radical. Indeed, lipid peroxidation and hemolysis in RBC from vitamin E-deficient rats were concurrent rather than consecutive events. Phase contrast microscopy showed that rigid, crenated RBC with a precipitate around the interior periphery formed during glutathione-induced oxidant stress. The precipitate dissolved slowly as the crenated RBC were converted to smooth ghosts. It appeared that protein precipitates involving mixed disulfide bonds were reduced and solubilized when extracellular glutathione penetrated the ruptured cell. Comparisons between normal RBC and vitamin E-deficient RBC suggest that vitamin E has little effect on the inward diffusion of extra-cellular H(2)O(2). Vitamin E apparently interacts with different oxidant species derived from intracellular H(2)O(2) in preventing lipid peroxidation and the sulfhydryl group oxidation leading to hemolysis.     

New immunolatex spheres: visual markers of antigens on lymphocytes for scanning electron microscopy

New immunochemical reagents consisting of antibodies bound to small latex spheres were used as visual markers for the detection and localization of cell surface antigens by scanning electron microscopy. Cross-linked latex spheres of various sizes from 300 to 3,4000 A in diameter were synthesized by aqueous emulsion copolymerization of methacrylate derivatives containing hydroxyl and carboxyl functional groups. Proteins and other molecules containing primary amino groups were covalently bonded to the acrylic spheres under a variety of mild conditions by the aqueous carbodiimide, cyanogen bromide, and glutaraldehyde methods. For use in the indirect immunochemical-labeling technique, goat antibodies directed against rabbit immunoglobulins were bonded to the spheres. These immunolatex reagents were shown to bind only to cells (red blood and lymphocytes) which had previously been sensitized with rabbit antibodies against cell surface antigens. Mouse spleen lymphocytes with exposed immunoglobulins on their surface (B cells) were labeled with these spheres and distinguished from unlabeled or T lymphocytes by scanning electron microscopy. The distribution of Ig receptors on lymphocytes was also studied using the spheres as visual markers. When lymphocytes were fixed with glutaraldehyde and subsequently labeled with the immunolatex reagents, a random distribution was observed by scanning electron microscopy; a patchy distribution was observed when unfixed lymphocytes were used. These results are consistent with studies using ferritin-labeled antibodies (S. De Petris and M. Raff. 1973. Nature [Lond.]. 241:257.) and support the view that Ig receptors on lymphocytes undergo translational diffusion. In addition to serving as visual markers for scanning electron microscopy, these latex spheres tagged with fluorescent or radioactive molecules have applications as highly sensitive markers for fluorescent microscopy and as reagents for quantitative studies of cell surface antigens and other receptors.     

Transport of sugars into microvessels isolated from rat brain: a model for the blood-brain barrier.

Abstract— Microvessels (primarily capillaries) were isolated from the brains of rats 25-35 days of age. This preparation was characterized by light, transmission, and scanning electron microscopy. Transmission electron microscopy revealed that the endothelial cell membranes were intact and were impermeable to horseradish peroxidase. However, scanning electron microscopy revealed that damage to the membrane occurred during isolation. The isolated microvessel preparations were metabolically competent as demonstrated by their ability to metabolize [¹⁴C]glucose. Aliquots of microvessel preparation were incubated with radioactive non-metabolizable analogs of D-glucose at various concentrations. The kinetics of accumulation of radioactivity in the capillaries were analyzed according to a model for carrier-mediated diffusion and affinity constants for 3-O-methyl- D-glucose and 2-deoxyglucose were calculated (about 18 mM at 20°C in each case). These affinity constants are somewhat greater than that expected from whole animal experiments reported by other laboratories. This discrepancy is probably accounted for by the presence of a passive diffusion component. However, despite this complication, the primary mechanism for entry of D-glucose analogues at physiological concentrations is compatible with carrier-mediated transport since: the uptake of sugar analogs was shown to be saturable, to exhibit competition for uptake between structurally similar molecules, and to be non-concentrative. In contrast, the uptake of glycerol, mannitol, and L-glucose by isolated microvessels obeyed the kinetics of simple passive diffusion and was not saturable. Our results are compatible with the concept that the capillary is the anatomic locus of the blood-brain barrier and that this structure contains the carrier-mediated transport system for monosaccharide penetration into brain.     

Biorecognition through layer-by-layer polyelectrolyte assembly: in-situ hybridization on living cells

Encapsulated cells were formed from the assembly of cationic and anionic alternating layers using a number of polyelectrolyte-based systems. Chitosan, alginate, hyaluronic acid, and oligonucleotides were used as polyelectrolytes to encapsulate individual E. coli cells, which were used as a model. Zeta potential measurements taken for both chitosan/alginate and chitosan/hyaluronic acid systems indicate successful layer-by-layer (LbL) deposition and gave full reversal of the surface change eight times. Layer adsorption was further observed by fluorescence microscopy, and, through a newly developed protocol for sample preparation, transmission electron microscopy micrographs clearly showed the presence of LbL assembly on the outer layer of the cell membrane, in the nanometer range. A second generation of E. coli cells could be grown from encapsulated first generation cells, demonstrating that the cellular activity was not affected by the presence of polyelectrolyte multilayers. Hybridization between attached oligonucleotide sequences and the complementary sequence was demonstrated by both fluorescence spectroscopy and microscopy. Fluorescence energy transfer data recorded after hybrid formation showed that at a molar ratio of 10:20 (donor:acceptor), Q and I were 92.3% and 52.5%, respectively, which suggests that fluorescein fluorescence was quenched by 92.3% and that the fluorescence of rhodamine was enhanced by 52.5%. Oligonucleotide incorporation was stabilized by deposition of four alternating layers, hence offering not only the potential use of the encapsulated cell as a bio-recognition system but also its application in a number of fields such as oligonucleotide delivery, gene therapy, and the use of DNA as an immunocompatible coating.     

Spin label study of erythrocyte deformability I. Electron spin resonance spectral change under shear flow

A spin labeling method in electron spin resonance spectroscopy (ESR) is applied for the first time to study the deformability of human red blood cells (RBC). ESR measurements of a RBC suspension incubated with a fatty acid spin label were performed, using a narrow-gap flat ESR sample cell under various flow shear stresses (tau). Remarkable changes were observed in ESR spectra with tau, indicating that RBC are oriented in such a way that the greater part of the membrane surface is aligned parallel to the ESR cell walls. The diamide-treated, hardened RBC, in which the biconcave discoid shape remains intact under no shear stress, exhibit a smaller ESR spectral change with tau than the intact, demonstrating that the present method can be used to assess the deformation of RBC occurring with flow orientation. In particular, the relative amplitude of an ESR difference spectrum may be used as a measure of the elongation of RBC. The conclusion is further supported by experiments using glutaraldehyde-treated or heat-denatured RBC. All these ESR results are in good agreement with the corresponding results obtained by several different methods. The present spin labeling technique is thus proven to be applicable for evaluating RBC deformability.     

Multi-level communication of human retinal pigment epithelial cells via tunneling nanotubes

Background

Tunneling nanotubes (TNTs) may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE) cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis.

Methodology and Principal Findings

Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca²⁺ imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells.

Conclusions and Significance

Our observations indicate that human RPE cell line ARPE-19 cells communicate by tunneling nanotubes and can support different types of intercellular traffic.     

Multiple three‐dimensional mammalian cell aggregates formed away from solid substrata in ultrasound standing waves

Single and multiple three‐dimensional cell aggregates of human red blood cells (RBCs) and HepG2 cells were formed rapidly in low mega‐Hertz ultrasound standing wave fields of different geometries. A single discoid aggregate was formed in a half‐wavelength pathlength resonator at a cell concentration sufficient to produce a 3D structure. Multiple cell aggregates were formed on the axis of a cylindrical resonator with a plane transducer (discoid aggregates); in a resonator with a tubular transducer and in the cross‐fields of plane and tubular transducers and two plane orthogonal transducers (all cylindrical aggregates). Mechanically strong RBC aggregates were obtained by crosslinking with wheat germ agglutinin (WGA, a lectin). Scanning electron microscopy showed aggregate surface porous structures when RBCs were mixed with WGA before sonication and tighter packing when ultrasonically preformed aggregates were subsequently exposed to a flow containing WGA. HepG2 cell aggregates showed strong accumulation of F‐actin at sites of cell–cell contact consistent with increased mechanical stability. The aggregates had a porous surface, and yet confocal microscopy revealed a tight packing of cells in the aggregate's inner core. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Electron microscopic and hydrodynamic studies of protein A-immunoglobulin G soluble complexes

The soluble complexes formed by reacting staphylococcal protein A (SpA) with rabbit immunoglobulin G (IgG) antibodies were characterized by hydrodynamic and electron microscopic methods. In moderate SpA excess, equilibrium mixtures of SpA and rabbit IgG formed four discrete complexes that sedimented at approximately 7, 10, 13, and 15S. The putative complexes were visible by electron microscopy and appeared to contain one, two, three, and approximately five molecules of IgG. Probably because of its elongated shape, SpA was not clearly visible in these mixtures or in control preparations of SpA alone. Both native IgG and IgG modified by cleavage of its single-hinge disulfide bond formed similar complexes on interaction with SpA. It was possible to resolve heterogeneous mixtures of IgG-SpA complexes by using an analytical ultracentrifuge equipped with a photoelectric scanner interfaced to a small computer. The relative concentrations and sedimentation velocities of different complexes in a mixture were determined from computer-generated integral and derivative plots. Both hydrodynamic and electron microscopic methods revealed that the distribution of complexes was sensitive to the IgG to SpA molar ratio. The relative amounts of faster complexes increased as the IgG to SpA molar ratio was increased. Surprisingly, when the IgG to SpA molar ratio was greater than or equal to 2, the complexes were converted into a unique 17S complex. This rather unprecedented transformation was reversible: the addition of excess SpA caused the dissociation of the 17S complex into a mixture of the 7, 10, 13, and 15S structures. The average translational diffusion coefficient of the 17S complex was 2.62 +/- 0.13 Ficks. In the electron microscope, the complex appeared to be exceptionally compact with an average diameter of 287 A. The stoichiometry of the 17S complex, together with sedimentation equilibrium, diffusion, and electron microscopic measurements, indicated that it is composed of four molecules of IgG and two molecules of SpA.     

Mast cell degranulation and its inhibition by an anti-allergic agent tranilast. An electron microscopic study

Degranulation of IgE-sensitized rat mast cells by antigen was studied quantitatively in vitro and in vivo by electron microscopy. The inhibition of this degranulation by an anti-allergic drug, N-(3,4-dimethoxycinnamoyl)anthranilic acid (Tranilast), was also examined both in vitro and in vivo. In the in vitro study using peritoneal mast cells, alteration of the granules, cavity formation by fusion of the perigranular membrane and granule discharge due to fusion of the cavity membrane with the cell membrane were observed and were accompanied by histamine release. Scanning electron microscopy disclosed the extrusion of smooth, round bodies from pores formed on the cell surface. In the in vivo study of passive cutaneous anaphylaxis (PCA), the characteristic features of mast cell degranulation were obvious 5 min after the injection of antigen; leakage of dye increased progressively from 5 to 30 min but was not found at 6 h. From quantitative analysis of the substructure of mast cells, it was demonstrated that degranulation of IgE-sensitized mast cell induced by antigen was achieved by sequential exocytosis both in vitro and in vivo. Tranilast inhibited these changes to a remarkable extent and it was concluded that the inhibition of mast cell degranulation by this drug might play an important role in anti-allergic treatment.     

Atomistic modeling of water diffusion in hydrolytic biomaterials

One of the most promising applications of hydrolytically degrading biomaterials is their use as drug release carriers. These uses, however, require that the degradation and diffusion of drug are reliably predicted, which is complex to achieve through present experimental methods. Atomistic modeling can help in the knowledge-based design of degrading biomaterials with tuned drug delivery properties, giving insights on the small molecules diffusivity at intermediate states of the degradation process. We present here an atomistic-based approach to investigate the diffusion of water (through which hydrolytic degradation occurs) in degrading bulk models of poly(lactic acid) or PLA. We determine the water diffusion coefficient for different swelling states of the polymeric matrix (from almost dry to pure water) and for different degrees of degradation. We show that water diffusivity is highly influenced by the swelling degree, while little or not influenced by the degradation state. This approach, giving water diffusivity for different states of the matrix, can be combined with diffusion-reaction analytical methods in order to predict the degradation path on longer time scales. Furthermore, atomistic approach can be used to investigate diffusion of other relevant small molecules, eventually leading to the a priori knowledge of degradable biomaterials transport properties, helping the design of the drug delivery systems.     

Internalized Chitosan Nanoparticles Persist for Long Time in Cultured Cells

Chitosan-based nanoparticles (chiNPs) are considered to be potentially good carriers for the sustained intracellular delivery of specific molecules. However, scarce attention has been paid to the long-lasting permanence of these NPs in the intracellular milieu, as well as to their intracellular fate (i.e., distribution, interaction with cell organelles, and degradation) in the long term. In the present study, the presence and subcellular location of FITC-labelled chiNPs were monitored in HeLa cells up to 14 days post-administration using multicolorfluorescence confocal microscopy and diaminobenzidine photo-oxidation at transmission electron microscopy. The main result of the present study is the demonstration that internalized chiNPs persist inside the cell up to two weeks, occurring in both the cytoplasm and nucleus; accordingly, chiNPs are able to pass from mother to daughter cells through several mitotic cycles. The cells did not show increased mortality or structural damage up to 14 days after chiNP exposure.     

Nanopipette delivery of individual molecules to cellular compartments for single-molecule fluorescence tracking

We have developed a new method, using a nanopipette, for controlled voltage-driven delivery of individual fluorescently labeled probe molecules to the plasma membrane which we used for single-molecule fluorescence tracking (SMT). The advantages of the method are 1), application of the probe to predefined regions on the membrane; 2), release of only one or a few molecules onto the cell surface; 3), when combined with total internal reflection fluorescence microscopy, very low background due to unbound molecules; and 4), the ability to first optimize the experiment and then repeat it on the same cell. We validated the method by performing an SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin in different surface domains of boar spermatozoa. We found little deviation from Brownian diffusion with a mean diffusion coefficient of 0.79 +/- 0.04 microm(2)/s in the acrosomal region and 0.10 +/- 0.02 microm(2)/s in the postacrosomal region; this difference probably reflects different membrane structures. We also showed that we can analyze diffusional properties of different subregions of the cell membrane and probe for the presence of diffusion barriers. It should be straightforward to extend this new method to other probes and cells, and it can be used as a new tool to investigate the cell membrane.     

Glial Cells in Abdominal Ganglia of Crayfish

Glial cells of abdominal ganglia of crayfish have been studied by transmission electron microscopy. Four cell types can be defined: (1) perivascular glial cells, close to the vascular spaces; (2) perineuronal glial cells, the processes of which ensheathe neuron perikarya; (3) adaxonal glial cells ensheathing axons; (4) neuropilar glial cells, associated with synapsing terminals in the neuropile. Neuropilar glia, adaxonal glia and the system formed by perineuronal and perivascular glia separate different functional zones of the neurons from the hemolymph or the electron dense extracellular matrix. These glial arrangements could play a similar role in hemato-neuronal transport. Gap-like junctions between glia and neuron cell bodies are frequent and could be involved in direct triggering of glial activities related to neurons.     

Diffusion chamber colony-forming unit (CFU-d): a primitive stem cell

Assay of hematopoietic precursor cells in diffusion chambers (DCs) implanted intraperitoneally in experimental animals provides a powerful tool for studying stem cell kinetics in vivo. In this system, the effect of cell migration (which complicates whole animal studies) is eliminated because the membranes utilized in the construction of the chambers are impermeable for cells, while permitting free passage of molecules present in the humoral phase of the host. As judged by light microscopy, conditions in the DC cultures primarily favor macrophage and granulocyte growth. However, the use of in vitro and in vivo subculture to further analyze chamber contents has demonstrated that the system supports proliferation of early hematopoietic progenitors. Additionally, cells capable of rescuing lethally irradiated mice proliferate in DC cultures. Development of the plasma clot DC technique has revealed that most of the growth occurs in colonies which are derived from single cells (CFU-d). Characterization of these cells indicates that they are at least as primitive as other colony-forming cells and, also based on subculture studies, can differentiate along several hematopoietic lineages. In addition to normal CFU-d, both embryonal and leukemic cells can give rise to granulocytes, macrophages, megakaryocytes and erythroid cells in the DC cultures. Evaluation of the effects of humoral factors on hematopoietic cell proliferation and differentiation in the system has led to the identification of both stimulators and inhibitors that may be different from the well-characterized cytokines. Thus, the system seems to be useful for detecting molecules controlling the most primitive stages of hematopoiesis. We believe that the DC culture technique holds enormous potential in the study of stem cell proliferation and differentiation in vivo.     

A stereologic approach to estimate the number of immunogold-labeled molecules in cells of tubules

OBJECTIVE: The quantity of molecules can be measured very precisely by molecular biological methods, but the capabilities of these are limited to measure only the total mass of tissue. For estimating the number of molecules at the cell level, it is necessary to combine an immunohistochemical protocol with designed-based principles of stereology at the level of electron microscopy (EM). This article focuses on the problems and practical solutions of fitting together immunohistochemistry, stereology, and electron microscopy for the estimation of the number of angiotensin II AT1 receptors in rat kidney arterioles. STUDY DESIGN: We performed the preembedding immunostaining of angiotensin II AT1 receptors using the silver-enhanced immunogold labeling system at EM level on serial sections of renal arterioles from 5 rats. RESULTS: Using this method the number of molecules can be estimated along the renal arterioles separately on the cell's surface, in cytoplasm, in nucleus, or in any subcellular location. CONCLUSION: For estimating the number of AT1 receptors, we designed a protocol that took into account the requirements for both immuno-EM and stereology. This method can be applied for estimating any molecule number in different types of cells in tubules.     

Serotonin-storing cells of the chicken duodenum: light,fluorescence and electron microscopy,and immunohistochemistry

Summary In an attempt to identify duodenal endocrine cells emitting formaldehyde-induced fluorescence (FIF), chicken duodena were studied by combined fluorescence, ultrastructural, silver impregnation and immunohistochemical methods in the same or consecutive sections. Our results show that: (1) Almost all the cells emitting yellow fluorescence by both the Falck-Hillarp and the Furness methods exhibit an immunohistochemical reaction with serotonin (5-HT) antiserum. (2) Almost all cells radiating yellow fluorescence by the Furness method stain with toluidine blue in Epon-embedded sections but, by high-voltage electron microscopy, can be subdivided into two types of cell containing either small round or polymorphous types of granules. (3) In the sections from which resin had been removed, all the cells emitting yellow FIF show argentaffinity by the Singh method, but not all cells display argyrophilia with the Grimelius method. (4) Cells exhibiting both argyrophil and argentaffin reactions in deresined serial sections are also separated into two types of cell, containing either small spherical or polymorphous types of granules by conventional electron microscopy in thin sections. Therefore, chicken enterochromaffin cells emit yellow FIF, store 5-HT, show both argentaffinity and argyrophilia, but are ultrastructurally classified into two types of granule-containing cells which may be related to polypeptides coexisting with 5-HT.