Immunogold Labeling Method Research Articles

Glycine-immunoreactive synaptic terminals in the nucleus tractus solitarii of the cat: ultrastructure and relationship to GABA-immunoreactive terminals.

Postembedding immunogold labeling methods applied to ultrathin and semithin sections of cat dorsomedial medulla showed that neuronal perikarya, dendrites, myelinated and nonmyelinated axons, and axon terminals in the nucleus tractus solitarii contain glycine immunoreactivity. Light microscopic observations on semithin sections revealed that these immunoreactive structures were unevenly distributed throughout the entire nucleus. At the electron microscopic level, synaptic terminals with high levels of glycine-immunoreactivity, assumed to represent those releasing glycine as a neurotransmitter, were discriminated from terminals containing low, probably metabolic levels of glycine-immunoreactivity, by a quantitative analysis method. This compared the immunolabeling of randomly sampled terminals with a reference level of labeling derived from sampling the perikarya of dorsal vagal neurones. The vast majority of these "glycinergic" terminals contained pleomorphic vesicles, formed symmetrical synaptic active zones, and targeted dendrites. They appeared to be more numerous in areas of the nucleus tractus solitarii adjoining the tractus solitarius, but rather scarce caudally, medially, ventrally, and in the dorsal motor vagal nucleus. In a random analysis of the entire nucleus tractus solitarii, 26.2% of sampled terminals were found to qualify as glycine-immunoreactive. In contrast, boutons immunoreactive for gamma-aminobutyric acid (GABA) were more evenly distributed throughout the dorsal vagal complex and accounted for 33.7% of the synaptic terminals sampled. A comparison of serial ultrathin sections suggested three subpopulations of synaptic terminals: one containing high levels of both GABA- and glycine-immunoreactivities (21% of all terminals sampled), one containing only GABA-immunoreactivity (12.7%), and relatively few terminals (5.2%) that were immunoreactive for glycine alone. These results were confirmed by dual labeling of sections using gold particles of different sizes. This study reports the first analysis of the ultrastructure of glycinergic nerve terminals in the cat dorsal vagal complex, and the pattern of coexistence of glycine and GABA observed provides an anatomical explanation for our previously reported inhibitory effects of glycine and GABA on neurones with cardiovascular and respiratory functions in the nucleus tractus solitarii.

Subcellular Distribution Of Basic Fibroblast Growth Factor-Like Molecules In Fibroblasts And Extracellular Matrix

Abstract Basic fibroblast growth factor (bFGF) is thought to play an important role in normal tissue repair and wound healing. It is a potent mitogenic and chemotactic factor for fibroblasts. To some degree bFGF regulates proliferation and extracellular matrix (ECM) production by these cells. In this study we investigate (i) the distribution of bFGF in fibroblasts and ECM of supporting tissues (ii) the subcellular location of this protein in vivo and, (iii) mechanisms for release of this growth factor from fibroblasts. Spurr's resin embedded ultrathin sections from adult rat sciatic nerve, adrenal gland, kidney; and from normal human lung and skin were labeled with either polyclonal (F3393-Sigma) or monoclonal antibodies (F6162-Sigma, C3316-ZymoGenetics Inc.) specific for bFGF using a two-step immunogold labeling method. Controls were incubated with either an equivalent volume of blocking solution (omitting the primary antibody) or an irrelevant antibody (Factor VIII, anti-Histamine)(Fig. 4).

Membrane skeleton spectrin in pollen and pollen tube

Immuno-western blots, immunofluorescence and immuno-gold labeling methods were used to study the existence and distribution of a homologue of the membrane skeleton spectrin in the pollen and pollen tube ofLilium davidii Duch. A spectrin homologue was found to exist in the pollen and pollen tube, distributed on membranes of secretory vesicles near Golgi apparatus.

Electron Microscopic Immunolocalization of Basic Fibroblast Growth Factor-Like Molecules in Capillary Endothelial Cells

Abstract Basic fibroblast growth factor (bFGF) is a potent angiogenic polypeptide. It promotes angiogenesis in vivo and in vitro by stimulating migration, proliferation and proteolytic activity of endothelial cells. Whereas several effects of exogenous bFGF on endothelial cells have been described, it has remained unclear how endogenous bFGF produced by vascular endothelial cells regulate angiogenesis. To further investigate functional implications of the distribution of bFGF, we undertook the present study. Our aims were (i) to identify the specific location of bFGF in endothelial cells using electron microscopy immunogold labeling technique (ii) to determine the distribution of bFGF in capillaries of different types of tissues. Tissue samples from sciatic nerve, hippocampus, adrenal gland and kidney of normal adult rats were fixed in 4% paraformaldehyde/1 to 5% glutaraldehyde and embedded in Spurr's resin. Ultrathin sections were labeled with either polyclonal (F3393-Sigma) or monoclonal antibodies (F6162-Sigma, C3316-ZymoGenetics) specific for bFGF using a two-step immunogold labeling method.

Ultrastructural Distribution of Basic Fibroblast Growth Factor-Like Molecules in Peripheral Nerves

Basic Fibroblast Growth Factor (bFGF) is a multifunctional polypeptide which has been shown to play a pivotal role in the survival and differentiation of nerve cells. Several trophic and non-trophic functions of this protein have been suggested in peripheral nerves. In spite of ample information about the distribution and effects of bFGF in central nervous system, few data are available concerning the localization of this protein in peripheral nerves. In view of the role of bFGF in regulation of trophic and non-trophic functions, we particularly focused on the presence and precise location of bFGF in peripheral nerves at the electron microscope level.Spurr's resin embedded ultrathin sections from adult rats’ sural nerves were labeled with either polyclonal (F3393-Sigma) or monoclonal antibodies (F6162-Sigma, C3316-Zymogenetics) specific for bFGF using two-step immunogold labeling method. Control samples were treated with either an equivalent volume of blocking solution (omitting the primary antibody) or an irrelevant antibody (Factor VIII, VGF, anti-histamine, anti-fibroblast 5B5).

Angiotensinogen, prorenin, and renin are Co-localized in the secretory granules of all glandular cells of the rat anterior pituitary: an immunoultrastructural study.

In addition to the circulating renin-angiotensin system (RAS), a local system has been postulated in the anterior pituitary because immunodetection of its components in various mammalian species. However, different cell types appear to be involved in different species, and there is no general consensus on the subcellular localization of prorenin, renin and angiotensinogen. In this ultrastructural study, we investigated and quantified the presence of these components using double or triple immunogold labeling methods, in all the immunologically identified glandular cell types of the rat anterior pituitary. In contrast to previous reports, all these components were identified not only in lactotropes and gonadotropes but also in somatotropes, corticotropes, and thyrotropes. The highest levels were detected in lactotropes and gonadotropes, and renin gave the greatest signal. Angiotensinogen, prorenin, and renin were co-localized in the secretory granules of all rat pituitary glandular cell types. The simultaneous detection of the substrate (angiotensinogen) and both its specific cleavage enzyme and its proenzyme within the same granule suggests intragranular processing of this component. Moreover, the localization of these three constituents in the secretory granules also suggests that, in the rat anterior pituitary, they follow the regulated secretory pathway.

Relationship of substance P- and CGRP-immunoreactive central endings of the primary afferent neurons to GABAergic interneurons in the guinea pig substantia gelatinosa.

The synaptic relationships between primary afferent central endings containing substance P (SP) and calcitonin gene-related peptide (CGRP) and GABAergic interneurons in the guinea pig substantia gelatinosa of the lumbar spinal dorsal horn were studied. The pre-embedding PAP method was used for detection of GABA and the post-embedding double immunogold labeling method for SP and CGRP detection. Immunogold particles specific for SP and CGRP were mainly localized separately or together in large synaptic vesicles devoid of dense cores. SP and CGRP immunoreactivity was separate or co-localized in small roundish, slender, sinuous or large scalloped (fan-like) terminals with closely packed round agranular synaptic vesicles of various sizes and few large dense core vesicles and mitochondria, which are thought to be capsaicin-sensitive primary afferent terminals. These SP- and CGRP-immunoreactive boutons make presynaptic or symmetric contacts with GABAergic dendrites and soma. These findings suggest that the central endings of nociceptive primary afferents transmit pain stimuli to intrinsic inhibitory interneurons, thereby modulating nociceptive information via a postsynaptic circuit.

Topographic Distribution of CD18 Integrin on Human Neutrophils as Related to Shape Changes and Movement Induced by Chemotactic Peptide and Phorbol Esters

The acquisition of high-affinity ligand binding may result from a topographical redistribution of β2 integrins (CD11/CD18) in the plane of the membrane in response to agonist-induced neutrophil stimulation. We examined the topographical distribution of CD18 on human neutrophils in relation with shapes changes and movements induced by stimulation with 10−8M N-formylmethionyl-leucyl-phenylalanine (fMLP) and 10−7Mphorbol myristate acetate (PMA). To localize CD18, we used immunogold-labeling methods and backscattered electron images obtained with scanning electron microscopy. On unstimulated neutrophils, CD18 integrin was randomly distributed on the nonvillous planar cell body. Stimulation of neutrophils with 10−8MfMLP and 10−7MPMA for 10 min induced distinctive shape changes, i.e., polar and nonpolar ruffled shapes, and upregulation of the surface membrane content of CD18. These changes were accompanied by a specific topographic distribution of CD18. fMLP-stimulated (10−8M) cells accumulated CD18 on the ruffled plasma membrane at the frontal pole of polar neutrophils. PMA-stimulated (10−7M) cells displayed CD18 aggregates on all plasma membrane domains, mainly on ruffles of nonpolar ruffled neutrophils. We conclude that the motile neutrophil responses elicited by chemotactic peptide and phorbol ester are associated with distinct patterns of topographic distribution of CD18 integrin. These features of CD18 expression may influence adhesive interactions mediated by human neutrophils.

The synaptic interrelationships between primary afferent terminals, cuneothalamic relay neurons and GABA-immunoreactive boutons in the rat cuneate nucleus

The present study described an ultrastructural synaptic configurations between primary afferent terminals (PATs), cuneothalamic relay neurons (CTNs) and GABA-immunoreactive (GABA-IR) boutons in the cuneate nucleus of rats using cervicothoracic dorsal rhizotomies, retrograde transport of wheat germ agglutinin conjugated with horseradish peroxidase complex (WGA-HRP) and anti-GABA immunogold labelling methods. With this procedure, direct synaptic relationships between the PATs, CTNs and GABA-IR boutons have been demonstrated. The most remarkable feature was the observation whereby an immunogold-labelled GABA-IR bouton was presynaptic to a WGA-HRP labelled dendrite of CTN and a degenerating PAT; the same PAT was in turn presynaptic to the HRP-labelled dendrite. This was evident in ten out of a total of 133 synaptic configurations that were closely scrutinized. Results of this study support the concept that GABA-IR boutons are not only involved in presynaptic inhibition on the primary afferent input to the cuneothalamic relay neurons, but also exert a simultaneous postsynaptic inhibition on these cells.

Ultrastructural immunogold labeling of lipid‐laden enterocytes from patients with genetic malabsorption syndromes

Intestinal biopsies from patients having genetic disorders of lipoprotein assembly and secretion, such as abetalipoproteinemia (ABL) or Anderson's disease (AD), contain large amounts of lipids which are accumulated in the enterocytes. Determination of the intracellular sites in which the lipids accumulate and to which apolipoproteins the lipids are bound would help to identify the defects in these diseases and further elucidate the mechanisms by which lipoprotein assembly and secretion occur normally. Ultrastructural immunogold labeling, however, is hampered by the poor preservation of the lipids accumulated in the enterocytes of these patients. We have used routine electron microscopy (fixation and ultra-thin sectioning) along with three methods for immunogold labeling of lipid-laden enterocytes: ultrathin cryosectioning, low temperature freeze substitution with embedding in Lowicryl K4M, and ultra-low temperature freeze substitution with embedding in Lowicryl HM20, to establish a protocol for investigating the intestinal tissue from these patients. Ultracryosectioning, while preserving the overall morphology of the lipid laden enterocytes, did not preserve the lipid content and the immunogold labeling of apolipoprotein B (ApoB) appeared dislocated. Freeze substitution and low temperature embedding in Lowicryl K4M, in contrast, appeared to better preserve the lipid and lipoprotein structures; however, the antigenicity of both apoAI and apoB appeared to be lost and no specific labeling could be obtained. Freeze substitution and embedding in Lowicryl HM20 best preserved the lipid and lipoprotein structures while maintaining apoprotein antigenicity. In conclusion, immunogold labeling of apolipoproteins on lipid structures in the lipid-laden enterocytes of patients with ABL and AD is best obtained by freeze substitution and embedding in Lowicryl HM20.

Ultrastructural immunogold labeling of lipid-laden enterocytes from patients with genetic malabsorption syndromes

Intestinal biopsies from patients having genetic disorders of lipoprotein assembly and secretion, such as abetalipoproteinemia (ABL) or Anderson's disease (AD), contain large amounts of lipids which are accumulated in the enterocytes. Determination of the intracellular sites in which the lipids accumulate and to which apolipoproteins the lipids are bound would help to identify the defects in these diseases and further elucidate the mechanisms by which lipoprotein assembly and secretion occur normally. Ultrastructural immunogold labeling, however, is hampered by the poor preservation of the lipids accumulated in the enterocytes of these patients. We have used routine electron microscopy (fixation and ultra-thin sectioning) along with three methods for immunogold labeling of lipid-laden enterocytes; ultrathin cryosectioning, low temperature freeze substitution with embedding in Lowicryl K4M, and ultra-low temperature freeze substitution with embedding in Lowicryl HM20, to establish a protocol for investigating the intestinal tissue from these patients. Ultracryosectioning, while preserving the overall morphology of the lipid laden enterocytes, did not preserve the lipid content and the immunogold labeling of apolipoprotein B (ApoB) appeared dislocated. Freeze substitution and low temperature embedding in Lowicryl K4M, in contrast, appeared to better preserve the lipid and lipoprotein structures; however, the antigenicity of both apoAI and apoB appeared to be lost and no specific labeling could be obtained. Freeze substitution and embedding in Lowicryl HM20 best preserved the lipid and lipoprotein structures while maintaining apoprotein antigenicity. In conclusion, immunogold labeling of apolipoproteins on lipid structures in the lipid-laden enterocytes of patients with ABL and AD is best obtained by freeze substitution and embedding in Lowicryl HM20.

Differentiating small round cell sarcomas of the soft parts by an innovative immunogold labeling method: an ultrastructural study.

A new immunoelectron microscopy procedure was developed by remaking the fixed-frozen tissue specimens into LR White resin blocks suitable for postembedding colloidal gold immunolabeling, and used to examine 16 cases of small round cell soft tissue sarcomas. In rhabdomyosarcoma, ultrastructural double-immunogold staining demonstrated a coexpression of muscle specific actin and desmin in the same tumor cell. In both Ewing's sarcoma and peripheral neuroepithelioma, the heterogeneous expression of MIC2 gene product (p30/32MIC2) in each tumor cell was demonstrated as well. In peripheral neuroepithelioma, the colloidal gold immunolabeling for neurofilament demonstrated the intermediate filaments surrounding microtubules. The procedure for ultrastructural colloidal gold immunolabeling using fixed-frozen tissue is thus considered to be useful not only for tumor diagnosis, but also for investigating various subcellular structures.

Detection of immunoreactive atrial and brain natriuretic peptides in the equine atrium.

The distribution of immunoreactivity (IR) for cardiodilatin/atrial natriuretic peptide (CDD/ANP) and brain natriuretic peptide (BNP) was examined immunohistochemically and immuno-electron-microscopically in the equine atrium, using specific antibodies. In the immunohistochemical studies, IR-CDD/ANP and IR-pBNP-26 (porcine BNP-26 immunoreactivity) was detected in the cytoplasm of the auricular cardiocytes, but IR-hBNP-32 (human BNP-32 immunoreactivity) was not. The double immunogold labelling method for IR-hBNP-28 and IR-pBNP-26 revealed that gold particles of different sizes were located in the same secretory granules in the cardiocyte, but no gold particles for IR-hBNP-32 were detected. These results show that CDD/ANP and porcine BNP-like peptides are colocalized in the same secretory granules of the equine atrium. They suggest that the equine atrium secretes both CDD/ANP and BNP-like peptides.

Immunoelectron microscopic localization of galectin-3, an IgE binding protein, in human mast cells and basophils.

Galectin-3 is an endogenous soluble lectin within the family called galectins that bind beta-galactosides. Homologs of the protein isolated from different sources were previously designated as IgE-binding protein (epsilon BP), CBP35, CPB30, Mac-2, RL-29, RLL, L-29, and HL-29. All are now renamed galectin-3. This lectin is widely distributed in cells and tissues of mice, rats, dogs, hamsters, and humans. Light microscopic immunohistochemistry and ultrastructural immunogold labeling methods were used to determine the distribution of galectin-3 in human mast cells of several organs, in mast cells developed in vitro from human fetal liver cells, and in human peripheral blood basophils. Immunolabeling for the protein was observed in mast cells from all sources and in basophils. The lectin was detected in the nucleus and/or the cytoplasm. The nuclear labeling was over heterochromatin whereas euchromatin was unlabeled. Cytoplasmic labeling was concentrated over secretory granules. The intensity of staining generally was greater in mast cells of skin when compared with that of mast cells in other locations and with that of basophils. Studies have indicated that in mast cells galectin-3 may be involved in promoting their adhesion to basal laminae. In this study the localization of galectin-3 in the secretory granules of human mast cells and basophils suggests that these cells may release this lectin when activated to degranulate.

Ultrastructural antigenic localization in Paragonimus iloktsuenensis during developmental stage by immunogold labeling method

Antigenic localization in Paragonimus iloktsuenensis worm tissues (tegument, intestine and vitelline gland) in different developmental stages of 2 weeks, 3 weeks, 4 weeks, 5 weeks and 33 weeks from albino rats (Sprague-Dawley) infected with P. iloktsuenensis was observed by electron microscopy. These worm tissues of different developmental stage of P. iloktsuenensis was observed on electromicrograph by immunogold labeling method using P. iloktsuenensis infected rat serum of 10 weeks. Antigenic localization was demonstrated as labeling of gold particles in tissues on electronmicrograph. In tegument, gold particles were labeled on tegumental tissue, generally more numerous on secretory granules in tegumental syncytium 2 weeks than those on the other elder developmental stages, but there was a little variation in antigenicity according to individual worm tissue. In general, antigenicity in tegumental tissue was not strong (gold particles: 0.1-5/1 microns 2). In intestine, a large number of gold particles (15-18/1 microns 2) were labeled in intestinal epithelium. Gold particles were concentrated especially on secretory granules in cytoplasm, and gold particles were labeled not only in cytoplasmic protrusions, but also in intestinal luminal contents. Intensity of labeling of gold particles was not correlated with developmental stage of worms. In vitelline gland, a large number of gold particles were labeled on vitelline globules. The gold particles in vitelline globules (8-11/1 microns 2) were concentrated in protoplasm among segmental globules.

False-positive labeling of the haustorial cell wall of bean rust: a problem in immunogold labeling of thin sections

False-positive labeling was studied by immunogold-labeling methods. It was observed during an investigation of chitinase distribution in rust-infected bean leaves. Sixteen of the 23 antisera/immunoglobulins tested gave distinct and apparently specific labeling of the haustorial cell wall of the fungus, also with non-immune sera and non-related antisera: all of them were rabbit antisera or immunoglobulins. Only the three monoclonal and four of the rabbit sera (two non-immune and two antisera against plant viruses) showed weak or no reactions. The effect was demonstrated with antiserum against rubisco, which labeled the haustorical cell wall in rather high dilutions. There were no disturbing antibodies against fungi in the sera of the rabbits as suitable acetone powders were inefficient. The type of binding and which component of the cell wall is involved are unknown. It is speculated that there might be a similar binding effect as with protein A to certain mammal immunoglobulins. Blocking with normal goat serum reduced the effect but use of bovine serum albumin (BSA), gelatine and Tween 20 was ineffective. The localization experiment of chitinase is a case in point concerning the misinterpretation of the specificity of an immunogold-labeling test. Therefore, standard controls are by no means sufficient and the specificity has to be confirmed by multiple controls.

Nuclear calmodulin/62 kDa calmodulin-binding protein complexes in interphasic and mitotic cells

We report here that a 62 kDa calmodulin-binding protein (p62), recently identified in the nucleus of rat hepatocytes, neurons and glial cells, consists of four polypeptides showing pI values between 5.9 and 6.1. By using a DNA-binding overlay assay we found that the two most basic of the p62 polypeptides bind both single- and double-stranded DNA. The intranuclear distribution of calmodulin and p62 was analysed in hepatocytes and astrocyte precursor cells, and in proliferating and differentiated astrocytes in primary cultures by immunogold-labeling methods. In non-dividing cells nuclear calmodulin was mostly localized in heterochromatin although it was also present in euchromatin and nucleoli. A similar pattern was observed for p62, with the difference that it was not located in nucleoli. p62/calmodulin complexes, mainly located over heterochromatin domains were also observed in interphasic cells. These complexes remained associated with the nuclear matrix after in situ sequential extraction with nucleases and high-salt containing buffers. In dividing cells, both calmodulin and p62 were found distributed over all the mitotic chromosomes but the p62/calmodulin aggregates were disrupted. These results suggest a role for calmodulin and p62 in the condensation of the chromatin.

Immunoelectron Microscopic Localization of C-Terminus of 43-kDa Dystrophin-Associated Glycoprotein in Normal Human Skeletal Myofibers

Ultrastructural localization of the C-terminus of a 43-kDa dystrophin-associated glycoprotein (43-DAG) and its relation to dystrophin were investigated in normal human skeletal myofibers by using single and double immunogold labelling methods. The C-terminal end of 43-DAG antibody was localized ultrastructurally at the inside surface of muscle plasma membrane and the sarcoplasmic side of the plasma membrane invaginations. Double immunolabelling electron microscopy disclosed that signals of the C-terminal end of 43-DAG and dystrophin were often associated with each other and formed a doublet at the inside surface of plasma membranes in normal human skeletal myofibers.

Identification of a Lipid Transfer Protein as the Major Protein in the Surface Wax of Broccoli (Brassica oleracea) Leaves

Presence of proteins in the surface wax of plants has been noted but no protein has been identified heretofore. A 9-kDa protein was purified as the major protein from the surface wax of broccoli (Brassica oleracea var. italica) leaves and the N-terminal amino acid sequence was determined. A cDNA corresponding to the wax-associated protein, designated WAX9, was sequenced revealing an open reading frame that codes for an 11.9-kDa polypeptide of 118 amino acids including a 25-amino acid leader peptide. The amino acid sequence showed 40 to 50% identity with nonspecific lipid transfer proteins isolated from various plants. Northern blots showed that a wax9 transcript of 650 nucleotides was highly expressed in leaves, stems, and flower buds and only at a reduced level in open flowers and not in roots. The transcript level decreased as the leaf fully expanded. The results suggest that the wax9 gene is expressed at the highest level at the time of maximal synthesis of the surface wax. Immunogold labeling studies showed that the WAX9 protein was present mainly in the cell wall of the epidermis and mesophyll tissues as well as in the phloem. However, immunological measurements showed that 50 and 4% of the total WAX9 was in the surface wax in the young and old leaves, respectively. Since immunogold labeling method would involve the loss of the surface wax, it would not detect the WAX9 protein in the wax. Thus, the present results reveal for the first time that a major portion of lipid transfer protein is in the surface wax.

Amiloride-sensitive channels in marginal cells in the stria vascularis of the guinea pig cochlea

We examined marginal cells in stria vascularis for the presence of amiloride-sensitive Na + channels, a possible pathway for maintaining a low Na + concentration in the endolymph. Whole-cell voltage-clamp experiment shows that amiloride at 1 μM concentration reversibly reduces inward current more than outward current. Immunogold-labeling method shows that the luminal and lateral membrane have antigenic sites for these antibodies. These observations indicate the presence of amiloride-sensitive channels in the marginal cell. If amiloride-sensitive channels in the luminal membrane are highly selective to Na +, they could be an efficient pathway for Na + uptake from the endolymph. In the basolateral membrane, amiloride-sensitive Na + channels may make a relatively small contribution to the unusual resting potential.