Immunolabeling Techniques Research Articles

Immunoelectron microscopy of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) in the syncytiotrophoblast of human placenta, clarifying the nature of the So-called large dense bodies

The synthetic and secretory processes of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) were investigated by immunoelectron microscopy which included the use of the double immunolabeling technique. The syncytiotrophoblast contains three kinds of granules: Small-sized granules (SG), middle-sized granules (MG), and large dense bodies (LB). Immunoreactions of hCG are localized in the MG and LB, while those of hPL are in the SG and LB. The double immunolabeling method proved without a doubt that hCG and hPL do not co-exist in the same SG or MG, but they are co-localized in the LB. This means that hCG and hPL are separately sorted into different secretory granules in the Golgi apparatus, but the LB are a kind of cytolysosome which are involved in the uptake and storage of both hPL and hCG.

Localization of cholecystokinin-like peptide in neuroendocrine cells of mammalian lungs: a light and electron microscopic immunohistochemical study.

We report immunohistochemical localization of cholecystokinin (CCK)-like immunoreactivity at the light and electron microscopy (EM) level in pulmonary neuroendocrine (NE) cells of human and other mammals (monkey, rabbit, rat, hamster, pig, dog and lamb). In addition, immunolocalization of CCK-like peptide was compared with that of bombesin (predominant peptide in human lung) and serotonin (an amine found in NE cells of most species). While CCK-like and serotonin-like immunoreactivity were identified in both solitary NE cells and NE cell clusters (neuroepithelial bodies, NEB) of all species studied, bombesin-like immunoreactive NE cells were found in human and monkey lungs only. The distribution and intensity of immunostaining for CCK-like peptide varied between species with some showing relatively high levels of expression (e.g., monkey, piglet, dog and lamb), others intermediate (human, rabbit) or weak immunostaining (rat, hamster). At the EM level, CCK-like immunoreactivity was localized in dense-core vesicles (DCV), the expected site of peptide storage. Using a double immunolabeling technique, CCK and serotonin were colocalized in some, but not all DCV. The potential role of CCK in the lung (or for other pulmonary peptides) may include a variety of functions such as modulation of bronchial or vascular tone, growth factor-like and/or hormonal effects.

The tissue distribution of factor XIIIa positive cells.

Factor XIIIa has previously been used to identify a subgroup of dermal dendritic cells which co-express class II MHC antigens, intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function associated antigen-1 (LFA-1), leading to the suggestion that they play a central role in skin immune responses. To examine whether these cells may also be important constituents of other organs we have performed an immunohistochemical survey of normal tissue to identify the distribution of this factor XIIIa positive subgroup of dendritic cells in humans. Routine tissue sections from a range of organs were assayed for factor XIIIa expression using a standard peroxidase-antiperoxidase (PAP) immunolabelling technique. Dendritic, factor XIIIa positive cells were observed in all tissues studied, but were most numerous in skin and mucosal tissues (gastrointestinal tract, bladder). They were also observed associated with epithelial structures in lung and kidney, but were only rarely observed in liver, thyroid, testis and spleen. The distinctive distribution of these cells is consistent with an important role for them in immune responses at those sites.

Robert Feulgen Prize Lecture. Distribution and role of gap junctions in normal myocardium and human ischaemic heart disease.

In the heart, individual cardiac muscle cells are linked by gap junctions. These junctions form low resistance pathways along which the electrical impulse flows rapidly and repeatedly between all the cells of the myocardium, ensuring their synchronous contraction. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera were raised to three synthetic peptides, each matching different cytoplasmically exposed portions of the sequence of connexin43, the major gap-junctional protein reported in the heart. The specificity of each antiserum for the peptide to which it was raised was established by dot blotting. New methods were developed for isolating enriched fractions of gap junctions from whole heart and from dissociated adult myocytes, in which detergent-treatment and raising the temperature (potentially damaging steps in previously described techniques) are avoided. Analysis of these fractions by SDS-polyacrylamide gel electrophoresis revealed major bands at 43 kDa (matching the molecular mass of connexin43) and at 70 kDa. Western blot experiments using our antisera indicated that both the 43-kDa and the 70-kDa bands represent cardiac gap-junctional proteins. Pre-embedding immunogold labelling of isolated gap junctions and post-embedding immunogold labelling of Lowicryl-embedded whole tissue demonstrated the specific binding of the antibodies to ultrastructurally defined gap junctions. One antiserum (raised to residues 131-142) was found to be particularly effective for cytochemical labelling. Using this antiserum for immunofluorescence labelling in combination with confocal scanning laser microscopy enabled highly sensitive detection and three-dimensional mapping of gap junctions through thick slices of cardiac tissue. By means of the serial optical sectioning ability of the confocal microscope, images of the entire gap junction population of complete en face-viewed disks were reconstructed. These reconstructions reveal the presence of large junctions arranged as a peripheral ring around the disk, with smaller junctions in an interior zone: an arrangement that may facilitate efficient intercellular transfer of current. By applying our immunolabelling techniques to tissue from hearts removed from transplant patients with advanced ischaemic heart disease, we have demonstrated that gap junction distribution between myocytes at the border zone of healed infarcts is markedly disordered. This abnormality may contribute to the genesis of reentrant arrhythmias in ischaemic heart disease.

Ultrastructural double labelling using colloidal gold

Immunogold double labelling is an under-utilized technique that is effective in indicating the presence, distribution and relative abundance of antigens in cellular systems. Current processing, embedding and immunolabelling techniques allow double labelling to be carried out at room temperatures, using routine electron microscope laboratory equipment. This review compares the available methodologies and presents some applications of light and ultrastructural immunogold labelling. The subjects of controls, antibody selection and quantitation are also discussed.

Heterogeneous targets of dopamine synapses in monkey prefrontal cortex demonstrated by serial section electron microscopy: a laminar analysis using the silver-enhanced diaminobenzidine sulfide (SEDS) immunolabeling technique.

Dopamine projections to the cerebral cortex have been implicated in normal and pathological cognitive processes, notably, Parkinson's disease and schizophrenia. To help elucidate the function of these dopamine axons, they were characterized by serial section electron microscopy in individual layers of monkey prefrontal cortex. Dopamine immunoreactivity was visualized with a silver precipitation technique that allowed clear resolution of the internal structures and cell membranes of labeled axons. Apart from the occasional large microtubule-filled axon, dopamine axons were thin and varicose with many clear synaptic vesicles and fewer dense-core vesicles. With few exceptions, dopamine synapses were symmetric and quite small, seen in only one to three serial sections. A determination of the "synaptic incidence" showed that only 39% of labeled varicosities formed identifiable synapses. However, it is certain that some small synapses could not be visualized even in serial sections, and it is possible that the vast majority if not all varicosities form synapses. Except for one soma, dendritic spines and shafts were the recipients of dopamine synapses. Many postsynaptic shafts were small and spiny, indicating that they were distal pyramidal dendrites. However, some postsynaptic shafts especially in supragranular layers had distinctly nonpyramidal features. These lacked spines, had a high density of synaptic inputs, and often had a strikingly varicose morphology. The data suggest that the majority of dopamine synapses in all layers are on pyramidal cells, but that a significant fraction are on presumed GABAergic nonpyramidal cells.

Endothelin, vasopressin, and substance P like immunoreactivity in cultured and intact epithelium from rabbit trachea.

The control of airways reactivity is essential to our understanding of disease processes such as asthma. Many studies have examined the neural control of the airways, but more recently there has been evidence to show that the epithelium lining the airways may influence airways reactivity. Rabbit tracheal epithelial cells were dispersed with enzymes, grown in primary culture and fixed. Tissue from intact tracheas was also sampled and fixed. Localisation of the vasoactive substances endothelin-1, arginine-vasopressin, and substance P was investigated by immunolabelling techniques. Scattered immunolabelling to endothelin-1, arginine-vasopressin, and substance P was found throughout the cultures (with < 20% of cells staining positively to each antibody). At the ultrastructural level this immunoreactivity was found in the cytoplasmic matrix. In addition, immunoreactivity of intact tissue to endothelin-1, arginine-vasopressin, and substance P was examined and positively staining cells were found to be scattered through the epithelium. The presence of these vasoactive substances within the epithelium lining the airways supports the view that epithelial cells may provide an additional mechanism in the control of airways reactivity.

Notes on a light and electron microscopic double-labeling method combining anterograde tracing with Phaseolus vulgaris leucoagglutinin and retrograde tracing with cholera toxin subunit B

Investigations of monosynaptic connections in the central nervous system have been hindered by the lack of compatible markers that can be used at both light and electron microscopic levels. In attempts to determine synaptic contacts between fibers originating in the substantia nigra and neurons projecting to the spinal cord, we have developed a double immunolabeling technique using anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and retrograde transport of unconjugated cholera toxin subunit B (CTB). In this report, we describe technical modifications which consistently produced superior labeling together with adequate ultrastructural preservation of the tissue and discuss the advantages of the two tracers.

Current concepts in pediatric burn care: the biology of cultured epithelial autografts: an eight-year study in pediatric burn patients.

An 8-year histopathological study of skin regeneration and wound healing in 22 pediatric patients treated with cultured epithelial autografts (CEA) grafted to full-thickness burn wounds excised to muscle fascia is reported. Biopsies of CEA have been analyzed by light microscopic, immunohistochemical, morphometric, electron microscopic and ultrastructural immunolabelling techniques and compared to controls of meshed split-thickness autograft (MSTA) interstices at comparable times postgrafting. At transplantation, CEA are undifferentiated and lack both granular and cornified cell layers. By 6 days postgrafting, CEA differentiate all normal epidermal strata but lack rete ridges. De novo formation of a confluent basal lamina and mature hemidesmosomes is complete by about 3 weeks. Anchoring fibrils appear sparse and immature (as in MSTA controls) compared to normal skin until about 6-12 months. CEA develop rete ridges and a neodermis with normal stromal and vascular organization at about 6-12 months, whereas MSTA interstice controls do not. At 4-5 years, elastin expression is also observed in the CEA neodermis, completing the dermal regeneration process. Normal epidermal differentiation is maintained long-term. These long-term results indicate that CEA regenerate a stable normal epidermis and are capable of inducing dermal regeneration from wound bed connective tissue.

The use of silver-enhanced 1-nm gold probes for light and electron microscopic localization of intra- and extracellular antigens in skin.

We used colloidal gold (1-nm diameter) with silver enhancement, in conjunction with a low-temperature post-embedding immunolabeling technique, to localize several antigens in normal skin at both the light and the electron microscopic level within the same tissue blocks. Normal skin subjected to cyrofixation and cryosubstitution and embedded in Lowicryl K11M was used as a substrate. Semi-thin sections (1 micron) were incubated in primary antibody (against epidermal basement membrane zone associated antigens and two keratin sub-types), biotinylated secondary antibodies, and then in 1-nm gold-conjugated streptavidin. Finally, the 1-nm gold label was enhanced using silver staining. Labeling of both basement membrane and keratin antigens was well demonstrated, and the area in the semi-thin sections showing the best structural preservation and the greatest intensity of immunolabeling was used to identify the part of the block to be used for ultra-thin sectioning. Ultra-thin sections were treated using a similar procedure to that employed for semi-thin sections. The labeling with silver-enhanced 1-nm gold probes was intense and readily visible by electron microscopy, even at low magnification. We have found this technique to have a high degree of specificity and sensitivity for labeling both intra- and extracellular antigens in skin, with the added advantage of providing the means for studies at both light microscopic and electron microscopic level.

Immunocytochemical localization of laccase L1 in wood decayed by Rigidoporus lignosus.

The cellular distribution of laccase L1 during degradation of wood chips by Rigidoporus lignosus, a tropical white rot fungus, was investigated by using anti-laccase L1 polyclonal antisera in conjunction with immunolabeling techniques. The enzyme was localized in the fungal cytoplasm and was associated with the plasmalemma and the fungal cell wall. An extracellular sheath, often observed around fungal cells, often contained laccase molecules. Diffusion of laccase within apparently unaltered wood was seldom observed. The enzyme penetrated all degraded cell walls, from the secondary wall toward the primary wall, including the middle lamella. Xylem cells showing advanced stages of decay were sometimes devoid of significant labeling. These data suggest that the initial attack on wood was not performed by laccase L1 of R. lignosus. Previous alteration of the lignocellulose complex may facilitate the movement of laccase within the wood cell walls. This immunogold study revealed that laccase localization during wood degradation seems limited not in space but in time.

Application of immunolabelling techniques to Bradyrhizobium dual occupation in lupinus nodules

This paper reports the immunoidentification of two Bradyrhizobium sp. (Lupinus) strains growing together in a culture medium or in nodules of Lupinus albus L. cv. Multolupa by ELISA and colloidal goldimmunolabelling techniques for observations at light and electron microscopy. The application of polyclonal IgG obtained against free-living bacteria has allowed immunoidentification of two strains in nodules with dual occupation, regardless of the bacterial development stages. No special distribution of the strains in these nodules was observed. Good immunolabelling was obtained with 5 mg L-1 IgG. Colloidal gold was on the bacterial cell wall. An immunolabelling on the bacteroid DNA fibrils appeared regardless of the strain that elicited the antiserum. Gold particles were not found on subcellular organelles or membranes of a plant nature, such as mitochondria, nucleus, Golgi apparatus, cell wall and peribacteroid membrane.

Presence of strong glucocorticoid receptor immunoreactivity within hypothalamic and hypophyseal cells containing pro-opiomelanocortic peptides

The presence of nuclear glucocorticoid receptor immunoreactivity (GR IR) was studied in the adrenocorticotropin (ACTH), β-Endorphin (β-END) and α-melanocyte stimulating hormone (α-MSH) IR neuronal populations of the rat hypothalamus and hypophysis using double immunolabelling techniques. All the nuclei of the ACTH/β-END/α-MSH IR neurons of the arcuate and periarcuate nuclei were strongly GR IR in the 48 h colchicine treated animal, but very few α-MSH-like IR perikarya located in the dorsal and lateral hypothalamus displayed nuclear GR IR. GR IR was present in the ACTH/β-END corticotrophs and absent in the intermediate lobe of the hypophysis. The data provide morphological evidence for a glucocorticoid action through a nuclear GR in the arcuate ACTH/β-END/ga-MSH IR neurons and the ACTH/β-END corticotrophs, whereas the α-MSH-like IR neurons of the lateral lateral hypothalamus and the melanotropes of the intermediate lobe may not be directly affected by glucocorticoids under normal conditions.

Synaptic associations between oxytocin-containing magnocellular neurons and neurons containing corticotropin-releasing factor in the rat magnocellular paraventricular nucleus

In the paraventricular nucleus (PVN) of the rat hypothalamus, we determined synaptic associations between oxytocin (OXT)-containing magnocellular neurons and parvocellular neurons containing corticotropin-releasing factor (CRF) by using a double immunolabeling technique in 7 animals. In single vibratome sections of the hypothalamus, immunoreactive CRF and OXT were labeled with silver-gold particles and diaminobenzidine (DAB) chromogen, respectively. By light microscopy CRF-containing fibers appeared to be black dots, some of which encircled magnocellular perikarya labeled with brown DAB chromogen in the PVN. By electron microscopy we discriminated OXT neurons having fine DAB-chromogen throughout the cytoplasm and on large secretory granules from CRF neurons having dense coarse particles of silver-gold. Occasional CRF axons terminated on perikarya or dendritic processes of OXT neurons, making synaptic contacts. The terminals which were characterized by having clusters of small clear vesicles and a few dense core vesicles showed equal thickenings of pre- and postsynaptic membranes at the synaptic junctions.

Gamma-aminobutyric acid immunoreactivity in the enterochromaffin cells of the rat stomach.

The present immunocytochemical study revealed gamma-aminobutyric acid (GABA) immunoreactivity in the oxyntic and pyloric mucosa of the rat stomach at light- and electron-microscopic levels. GABA-immunoreactive endocrine cells were numerously seen in the lower half portion of the pyloric mucosa but rarely in the oxyntic mucosa. These cells were round or oval in shape and sometimes had a short cytoplasmic process. Serotonin-immunoreactive enterochromaffin (EC) cells were also observed in the oxyntic and pyloric mucosa of the stomach. The distribution and shapes of the immunoreactive cells were similar to those of the GABA-immunoreactive cells. With a double immunolabeling technique using anti-GABA and antiserotonin serum, GABA-immunoreactive endocrine cells showed serotonin immunoreactivity and were identified as EC cells. At the electron-microscopic level the GABA-immunoreactive cells contained round or oval, spindle-like, pear-shaped granules in EC cells. The immunoreaction product in the EC cells was generally confined to the granular cores. These findings suggest that GABA may be synthesized in the EC cells and be released from the granules of the cells after adequate stimuli.

Ultrastructural immunolabeling: a general overview of techniques and applications.

Ultrastructural immunolabeling techniques combine the advantages of routine electron microscopy and detection of antigenic epitopes, the latter of which is customarily done by immunocytochemistry at the light microscopic level. In surgical pathology, immunocytochemistry has become routine to approach the differential diagnosis of difficult cases. Immunoelectron microscopy has been used primarily for research purposes or for addressing specific questions in diagnostic pathology. Ultrastructural immunolabeling is extremely useful in those instances in which the ultrastructural and immunocytochemical findings are not pathognomonic and are subject to interpretation. Preembedding and postembedding labeling techniques have been described in the literature. Preembedding techniques are not as applicable to diagnostic work, however. Their use remains rather limited to applications in which the antigen to be labeled cannot maintain its viability when exposed to fixatives. Even in such cases a new methodology--the LifeCell process--has emerged; this technique cryofixes tissues, thereby maintaining antigenic integrity. After cryofixation, a postembedding labeling technique can be utilized. Immunogold and peroxidase methods are used for labeling. Immunogold methods elegantly mark reaction sites with preservation of underlying morphology. Postembedding immunogold methods are used by most individuals working in the field. Ultrastructural labeling techniques are rapidly moving from classification as exclusively research tools to the diagnostic arena.

Visualization of a luteovirus in the vector aphid's body by two gold immunolabelling techniques: A comparative study

Two gold immunolabelling techniques using electron microscopy were compared to examine the in situ localization of a luteovirus, potato leafroll virus (PLRV), inside its main aphid vector, Myzus persicae SULZ. With Gildow's technique, virus particles were labelled prior to fixation, embedding by injecting PLRV-specific IgGs into the living aphids. This facilitated the detection of extracellular particles located between the basal lamina and plasmalemma by trapping them in aggregates. The heavy coating of particles by antibodies and gold indicated good labelling sensitivity. Isometric virus-like particles were also observed inside the cytoplasm, but they were non decorated because the cell membrane prevented labelling reagents from entering the cell. With the second technique, ultrathin sections were immunolabelled after fixation-embedding. Since PLRV lost its antigenicity when aphid tissues were normally treated for electron microscopy, the successful application of this technique required fixation in 4% formaldehyde before embedding in Lowicryl at low temperature; it was also necessary to use PLRV-specific monoclonal antibodies to eliminate non-specific reactions. In these conditions, all intra- and extra-cytoplasmic virions present on the surface of sections were surrounded by gold particles, but the antibody coating was not discernible, and, because the resin limited the access of markers to antigens, the inner virus particles were not labelled. In conclusion, both techniques must be applied on the same material to give complementary information.

CD11/CD18 cell surface adhesion glycoproteins: Discordance of monocyte function and expression in response to stimulation

The adherence of blood leukocytes to vascular endothelium precedes their diapedesis into the extravascular space. These processes require the expression of adherence glycoproteins on the cell surface of the leukocyte. The relative importance of these adherence molecules is so far poorly understood. However, there is evidence to suggest that a disparity exists between the surface receptor expression of these glycoproteins and leukocyte adherence to vascular endothelial cells in culture. We have investigated the importance of each of the adhesion glycoproteins CD11a, CD11b and CD11c in mediating the adherence of human monocytes to endothelial cells in culture. We have also investigated the chronological relationship between changes in monocyte adherence to endothelial cells and the surface expression of CD11a, CD11b, and CD11c following stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). The increase in adherence occurred within 1 minute, but declined if monocytes were preincubated with fMLP for up to 30 minutes. The surface expression of adherence molecules demonstrated a significant increase in CD11a and CD11b in the presence of fMLP after 10 min and was maintained while monocyte adherence to endothelium declined. These changes in surface receptor expression were quantitated using an immunolabeling technique. It is suggested that fMLP stimulation of monocyte adherence is unlikely to be solely dependent on increased surface receptor expression of adhesion molecules.

Identification of peroxidase-positive astrocytes by combined histochemical and immunolabeling techniques in situ and in cell culture.

A subpopulation of astrocytes in the vertebrate brain and in cysteamine-treated brain cell cultures contain cytoplasmic granules which exhibit an affinity for Gomori stains, orange-red autofluorescence, and non-enzymatic endogenous peroxidase activity. Visualization of these cells at the light microscopic level is confounded by the nonspecificity of the various histochemical methods routinely employed. In an attempt to circumvent this problem, we assayed for peroxidase-positive astrocytes using various combinations of diaminobenzidine (DAB) histochemistry and immunolabeling for the astrocyte-specific marker glial fibrillary acidic protein (GFAP). We determined that (a) DAB histochemistry in conjunction with avidin-biotin-immunoperoxidase labeling for GFAP specifically detects peroxidase-positive astrocytes in situ and (b) DAB histochemistry combined with indirect immunofluorescence for GFAP effectively demonstrates these cells in cysteamine-treated brain cell cultures.

Binding of nonspecific cross-reacting antigen, a granulocyte membrane glycoprotein, to Escherichia coli expressing type 1 fimbriae

Nonspecific cross-reacting antigen (NCA) is a well-characterized membrane glycoprotein on granulocytes, macrophages, and lung epithelium. Structural studies at the protein and genomic levels have revealed that NCA is a member of the immunoglobulin supergene family, and hybridization studies showed that the transcript level of NCA is induced by treatment with gamma interferon. These studies, as well as the expression of NCA on granulocytes, suggest a role for NCA in immune response. For a first step in studying this possible role, we have examined the binding of two glycoforms of NCA designated NCA-50 (Mr, 50,000) and TEX-75 (Mr, 75,000). Here we report the results from binding assays which demonstrate carbohydrate-mediated binding of Escherichia coli expressing type 1 fimbriae and of isolated type 1 fimbriae to NCA-50. TEX-75 did not bind to the purified fimbriae but bound slightly to the bacterial strain. Inhibition studies showed that the binding to NCA-50 involved interaction of mannose moieties on NCA-50 and lectins on the fimbriae. The binding of NCA-50 to bacterial fimbriae was confirmed by electron microscopy studies, using immunolabeling techniques. In addition, we show that the surface expression of NCA-50 (and presumably of other NCA species) on isolated polymorphonuclear leukocytes is increased following activation with the bacterial peptide formylmethionyl-leucyl-phenylalanine, consistent with a role for NCA in immune response.