Immunogold Labeling For Electron Microscopy Research Articles

Synaptic Transmission from Somatostatin-expressing Interneurons to Excitatory Neurons Mediated by α5-subunit-containing GABAA Receptors in the Developing Visual Cortex

Dendrite-targeting somatostatin-expressing interneurons (SST-INs) powerfully control signal integration and synaptic plasticity in pyramidal dendrites during cortical development. We previously showed that synaptic transmission from SST-INs to pyramidal cells (PCs) (SST-IN → PC) in the mouse visual cortex suddenly declined at around the second postnatal week. However, it is unclear what specific postsynaptic mechanisms underlie this developmental change. Using multiple whole-cell patch-clamp recordings, we found that application of an α5-GABAA receptor-selective inverse agonist, alpha5IA, significantly weakened SST-IN → PC unitary inhibitory postsynaptic currents (uIPSCs) in layer 2/3 of the mouse visual cortex, but had no effect on uIPSCs from SST-INs to other types of interneurons. The extent of alpha5IA-induced reduction of SST-IN → PC synaptic transmission was significantly larger at postnatal days 11–13 (P11–13) than P14–17. Moreover, α5-subunit-containing GABAA receptors (α5-GABAARs)-mediated uIPSCs had slow rise and decay kinetics. Apart from pharmacological test, we observed that SST-IN → PC synapses did indeed contain α5-GABAARs by immunogold labeling for electron microscopy. More importantly, coinciding with the weakening of SST-IN → PC synaptic transmission, the number of α5-GABAAR particles in SST-IN → PC synapses significantly decreased at around the second postnatal week. Together, these data indicate that α5-GABAARs are involved in synaptic transmission from SST-INs to PCs in the neocortex, and are significantly diminished around the second postnatal week.

Identification of L-ferritin in Neuromelanin Granules of the Human Substantia Nigra: A TARGETED PROTEOMICS APPROACH

In the pigmented dopaminergic neurons of the human substantia nigra pars compacta the system relevant in iron storage is the polymer neuromelanin (NM). Although in most cells this function is mainly accomplished by ferritin, this protein complex appears not to be expressed in NM-containing neurons. Nevertheless the conceivable presence of iron-storing proteins as part of the NM granules has recently been discussed on the basis of Mössbauer spectroscopy and synchrotron x-ray microspectroscopy. Intriguingly by combining subcellular fractionation of NM granules, peptide sequencing via tandem mass spectrometry, and the additional confirmation by multiple reaction monitoring and immunogold labeling for electron microscopy, L-ferritin could now be unambiguously identified and localized in NM granules for the first time. This finding not only supports direct evidence for a regulatory role of L-ferritin in neuroectodermal cell pigmentation but also integrates a new player within a complicated network governing iron homeostasis in the dopamine neurons of the human substantia nigra. Thus our finding entails far reaching implications especially when considering etiopathogenetic aspects of Parkinson disease.

Optimization of Immunogold Labeling TEM: An ELISA-based Method for Rapid and Convenient Simulation of Processing Conditions for Quantitative Detection of Antigen

We developed an ELISA-based method for rapid optimization of various tissue processing parameters in immunogold labeling for electron microscopy. The effects of aldehyde fixation, tannic acid, postfixation, dehydration, temperature, and antigen retrieval on antibody binding activity of Vitreoscilla hemoglobin (VHb) expressed in E. coli cells were assayed by ELISA and the results confirmed by quantitative immunogold labeling transmission electron microscopy (TEM). Our results demonstrated that low concentrations (0.2%) of glutaraldehyde fixation caused minimal loss in total binding compared to higher concentrations. Dehydration in up to 70% ethanol resulted in some distortion of cellular ultrastructure but better antibody binding activity compared to dehydration up to 100%. Postfixation or incorporation of tannic acid in the primary fixative caused almost total loss of activity, whereas antigen retrieval of osmium-postfixed material resulted in approximately 90-100% recovery. The sensitivity of detection of proteins by immunogold labeling electron microscopy depends on the retention of antibody binding activity during tissue processing steps, e.g., fixation and dehydration. Our study indicated that an ELISA-based screening method of various tissue processing procedures could help in rapid selection and optimization of a suitable protocol for immunogold localization and quantification of antigen by TEM.

Cholesterol-induced changes of type VIII collagen expression and distribution in carotid arteries of rabbit.

Lipoproteins play a major role in cardiovascular disease and atherosclerosis. In the vascular wall, they strongly influence the organization of extracellular matrix. The present study set out to investigate the changes in the extracellular matrix of the vessel wall induced by atherogenic diet, focusing on type VIII collagen, a vascular collagen that has not previously been investigated in detail. The influence of cholesterol diet on the expression, distribution, and deposition of type VIII collagen was examined in carotid arteries of New Zealand White rabbits. Carotid arteries of rabbits receiving diet supplemented with 1% cholesterol for 6 weeks and those on the same regimen followed by normal chow for 1 day, 10 days, 5 weeks, and 12 weeks were studied and compared with controls not exposed to the cholesterol diet. Carotid arteries of normocholesterolemic rabbits contained type VIII collagen-expressing cells in all layers, with focal accumulations of expressing cells in the subendothelial areas, the outer medial zone, and the adventitia. In response to cholesterol diet, type VIII collagen synthesis was reduced in media and adventitia and the distribution patterns changed. Expressing cells were found predominantly in the endothelium, and type VIII collagen accumulated in the intimal space. Immunogold labeling for electron microscopy revealed that type VIII collagen in the intima is associated with microfibrils extending from the internal elastic lamina. Withdrawal of cholesterol resulted in reestablishment of the normal distribution pattern. Northern and Western blot analyses supported the immunoconfocal and in situ hybridization data, demonstrating decreased type VIII collagen expression in response to cholesterol diet and progressive recovery to normal levels with time after withdrawal of cholesterol. Our study demonstrates that type VIII collagen is modulated in the presence of cholesterol. The data indicate that type VIII collagen is specifically remodeled during early experimental atherosclerosis, implying a role for this extracellular matrix component in neointimal growth.

Evidence for an alpha-granular pool of the cytoskeletal protein alpha-actinin in human platelets that redistributes with the adhesive glycoprotein thrombospondin-1 during the exocytotic process.

In a previous study, we have demonstrated that the platelet adhesive glycoprotein thrombospondin-1 (TSP-1) interacts specifically with the cytoskeletal protein alpha-actinin in a solid-phase binding assay. Stored in the alpha-granules of platelets, TSP-1 is secreted during cell activation and binds to the plasma membrane promoting the platelet macroaggregate formation. However, the molecular mechanism by which TSP-1 reaches and binds to the platelet surface is to date unelucidated. alpha-Actinin is an actin-binding and actinin-cross-linking protein that is present in most cells and may act as a link between the bundles of F-actin and the plasma membrane. In this study, we have investigated a possible interaction of alpha-actinin with TSP-1 in platelets by examining their respective subcellular location during the platelet activation process. By indirect immunofluorescence. alpha-actinin was found to display a granular staining in resting platelets similar to that of TSP-1. Performing postembedding immunogold labeling for electron microscopy, we detected the presence of alpha-actinin throughout the cytoplasm, but the strongest gold staining was found in organelles identified as alpha-granules on the basis of their ultrastructure and TSP-1 content. With the use of double immunogold labeling on platelets at different stages of activation by thrombin, both alpha-actinin and TSP-1 were seen redistributing from the alpha-granules to the platelet surface via the open canalicular system (OCS). At the same time, the cytoplasmic alpha-actinin concentrated toward the plasma membrane, but no colocalization with the F-actin bundles was evidenced. Finally, preembedding immunogold labeling and immunoprecipitation of 125I-surface-labeled, thrombin-activated platelets further demonstrated that alpha-actinin was expressed on the plasma membrane in the absence of any detectable expression of actin and that it could from molecular complexes with TSP-1 on activated platelets. These results suggest that alpha-actinin found to be present on the platelet surface together with TSP-1 originates in the alpha-granules by fusion of the alpha-granules with the plasma membrane during platelet exocytosis.

Localization of human chorionic gonadotropin and placental lactogen by immunogold labeling for electron microscopy: Technique and limitations

To demonstrate human chorionic gonadotropin (hCG) and human placental lactogen (hPL) secretory granules in placenta and to illustrate newer embedding techniques and specific immunospecificity problems in the placenta, labeling experiments using immunogold or peroxidase combined with avidin-biotin enhancement in epoxy LX-112-, Araldite-, or LR gold-embedded tissue fixed in 2.5% glutaraldehyde or 2.5% paraformaldehyde were carried out in term and first-trimester normal human placenta and in partial hydatidiform moles. Increased sensitivity of the low-temperature LR gold method was found for hPL-labeled granules. βhCG-labeled granules were noted in syncytium of first-trimester placenta, and βhCG-containing granules in hydatidiform moles were similar to those of normal placenta. Paraformaldehyde fixation and LR gold embedding permitted identification of endoplasmic reticulum-associated labeling not observed with other methods. A brief review and discussion of immunolabeling methods, controls, and embedding materials is presented. We conclude that further refinement of peptide localization methods in the placenta is possible but must take into account the abundant potentially cross-reacting peptides present in the placenta. Microsc. Res. Tech. 38:176–187, 1997. © 1997 Wiley-Liss, Inc.

Localization of human chorionic gonadoptropin and placental lactogen by immunogold labeling for electron microscopy: technique and limitations.

To demonstrate human chorionic gonadotropin (hCG) and human placental lactogen (hPL) secretory granules in placenta and to illustrate newer embedding techniques and specific immunospecificity problems in the placenta, labeling experiments using immunogold or peroxidase combined with avidin-biotin enhancement in epoxy LX-112-, Araldite-, or LR gold-embedded tissue fixed in 2.5% glutaraldehyde or 2.5% paraformaldehyde were carried out in term and first-trimester normal human placenta and in partial hydatidiform moles. Increased sensitivity of the low-temperature LR gold method was found for hPL-labeled granules. beta hCG-labeled granules were noted in syncytium of first-trimester placenta, and beta hCG-containing granules in hydatidiform moles were similar to those of normal placenta. Paraformaldehyde fixation and LR gold embedding permitted identification of endoplasmic reticulum-associated labeling not observed with other methods. A brief review and discussion of immunolabeling methods, controls, and embedding materials is presented. We conclude that further refinement of peptide localization methods in the placenta is possible but must take into account the abundant potentially cross-reacting peptides present in the placenta.

Immunological characterization of trichocyst proteins in the ciliate Pseudomicrothorax dubius.

Ejectable trichocysts were isolated from the ciliate Pseudomicrothorax dubius. Polyclonal antibodies were raised against three groups of trichocyst proteins: G1 (30-31 kDa), G2 (26-27 kDa) and G3 (15-20 kDa). By indirect immunofluorescence, the three antisera strongly label the shafts of ejected trichocysts and the proximal ends of condensed trichocysts within the cells. By immunogold labeling for electron microscopy, the three sera specifically recognize the shafts of both extended and condensed trichocysts and shaft precursors in pretrichocysts as well. On one-dimensional immunoblots of isolated trichocysts, anti-G1 serum recognizes the G1 proteins, anti-G2 serum detects G2 proteins and some G1 proteins, and anti-G3 serum reacts with 15 bands, mainly the G3 and (30-41)-kDa proteins. In cells with and without trichocysts, the sera recognize non-ejectable trichocyst proteins at 41-42 kDa and 47 kDa. On two-dimensional immunoblots of isolated trichocysts, anti-G1 serum labels proteins with a pI of 4.75-5.7, anti-G2 serum labels proteins with a pI of 4.75-6.25 and anti-G3 serum labels proteins with a pI of 4.7-6.6. Analyses of cells with and without trichocysts allow identification of possible precursors between 41 and 47 kDa. Some are in the same pI range as their putative products, but others, labeled by anti-G3 serum, are less acidic than most of their mature products.

Immunocytochemical evidence for a possible role of cross-linked keratinocyte envelopes in stratum corneum cohesion.

Cross-linked cornified envelopes are cell structures specifically synthesized by terminally differentiating keratinocytes. They are composed of proteins deposited at the cell periphery under the plasma membrane, and can be purified from epidermis by physicochemical extractions. The resulting keratinocyte "shells" are highly insoluble structures devoid of cytoplasmic components. The rigidity of the stratum corneum cell envelope seems to be one of the essential factors contributing to the physical resistance of this most superficial epidermal layer. We studied the purified cell envelopes from human plantar horny layer to determine their antigenic composition and protein distribution. The extraction protocol consisted of four 10-min cycles of boiling in 10 mM Tris-HCl buffer containing 2% SDS and 1% beta-mercaptoethanol. The absence of any extractable proteins persisting in the purified pellets was checked with SDS-PAGE of the sample electroeluates. Indirect immunofluorescence as well as pre- and post-embedding immunogold labeling for electron microscopy revealed the persistence of several keratinocyte antigenic determinants on the purified substrates. The antibodies directed against involucrin, keratin 10, desmoplakin I + II, desmoglein (intracellular epitope), intercellular corneodesmosome proteins, and filaggrin (a considerably weaker reactivity) labeled the cell envelopes according to the ultrastructural localization pattern characteristic for a given antigen. We conclude that the cytoskeletal and desmosomal components become "embedded" in the highly cross-linked cornified envelope structures during the process of keratinocyte terminal differentiation. This underlines the central role of cornified envelopes in the physical resistance of superficial epidermal layers and indicates a possible importance of junctional proteins in this function.

Identification of seven surface-exposed Brucella outer membrane proteins by use of monoclonal antibodies: immunogold labeling for electron microscopy and enzyme-linked immunosorbent assay

A panel of monoclonal antibodies (MAbs) to seven Brucella outer membrane proteins were characterized. These antibodies were obtained by immunizing mice with sodium dodecyl sulfate-insoluble (SDS-I) fractions, cell walls, or whole bacterial cells of Brucella abortus or B. melitensis. Enzyme-linked immunosorbent assays were used to screen the hybridoma supernatants and to determine their binding at the surface of rough and smooth B. abortus and B. melitensis cells. The outer membrane proteins (OMPs) recognized by these antibodies were the proteins with molecular masses of 25 to 27 kDa and 36 to 38 kDa (porin) (major proteins) and the proteins with molecular masses of 10, 16.5, 19, 31 to 34, and 89 kDa (minor proteins). Surface exposure of these OMPs was visualized by electron microscopy by using the MAbs and immunogold labeling. Binding of the MAbs on whole rough bacterial cells indicates that the 10-, 16.5-, 19-, 25- to 27-, 31- to 34-, 36- to 38-, and 89-kDa OMPs are exposed at the cell surface. However, enzyme-linked immunosorbent assay results indicate a much better binding of the anti-OMP MAbs on rough strains than on the corresponding smooth strains except for the anti-19-kDa MAb. Immunoelectron microscopy showed that on smooth B. abortus cells only the 89- and 31- to 34-kDa OMPs were not accessible to the MAbs tested. Binding of the anti-31- to 34-kDa MAb at the cell surface was observed for the rough B. abortus cells and for the rough and smooth B. melitensis cells. These results indicate the importance of steric hindrance due to the presence of the long lipopolysaccharide O side chains in the accessibility of OMPs on smooth Brucella strains and should be considered when undertaking vaccine development.

Immunogold Labeling for Electron Microscopy

Immunogold Labeling for Electron Microscopy

Induction of class I major histocompatibility complex antigen expression by West Nile virus on gamma interferon-refractory early murine trophoblast cells.

Primary murine trophoblast giant cells (TGC) do not express detectable major histocompatibility complex (MHC) antigens and are refractory to the MHC-increasing effects of alpha and beta (virus-induced) interferons and gamma (immune type) interferon during early implantation (postcoital days 3.5-6). West Nile virus infection of primary TGC monolayers from postcoital-day-3.5 preimplantation blastocysts induced paternal MHC antigen expression within 16 hr, as detected by immunogold labeling for electron microscopy. Induction is unlikely to have been mediated by secreted virus-induced interferons or other factors, as it occurred in the presence of high concentrations of anti-alpha/beta interferon antibodies and was not induced by virus-inactivated supernatants from MHC-induced primary TGC cultures. Attempts to induce MHC antigen expression with poly(I.C) or recombinant tumor necrosis factor alpha in primary TGC cultures also failed. Thus, the apparent inhibition of MHC antigen expression in primary TGC during early implantation and their refractoriness to induction of de novo MHC antigen expression is not absolute. This may represent a maternal-and/or species-protective evolutionary device. As such, manipulation of this phenomenon may allow a conclusive assessment of the significance of inhibition of MHC antigen expression on trophoblast cells in the implanting semiallogeneic embryo.