scholarly journals Usefulness of the immunogold technique in quantitation of a soluble protein in ultra-thin sections.

1987 ◽  
Vol 35 (4) ◽  
pp. 405-410 ◽  
Author(s):  
G Posthuma ◽  
J W Slot ◽  
H J Geuze
Keyword(s):  
Model System ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Embedded Model ◽  
Intracellular Compartments ◽  
Embedded Blocks ◽  
Gelatin Concentration ◽  
Lowicryl K4m

We used a model system to study whether measurements of absolute local antigen concentrations at the electron microscopic level are feasible by counting immunogold labeling density in ultra-thin sections. The model system consisted of a matrix of a variable concentration of gelatin, which was mixed with given concentrations of rat pancreas amylase and fixed according to various fixation protocols. With a relatively mild fixation, there was no clear proportionality between anti-amylase gold labeling and amylase concentration in ultra-thin cryosections. This was presumably due to uncontrolled loss of amylase from the sections. After stronger fixation with 2% glutaraldehyde for 4 hr, labeling density reflected the amylase concentration very well. We observed that matrix (gelatin) density influenced labeling density. A low gelatin concentration of 5% allowed penetration of immunoreagents into the cryosection, resulting in a high and variable labeling density. In gelatin concentrations of 10% and 20%, labeling density was lower but proportional to amylase concentration. To establish an equal (minimal) penetration of immunoreagents, we embedded model blocks with different matrix densities in polyacrylamide (PAA). In ultra-thin cryosections of these PAA-embedded blocks, anti-amylase labeling was proportional to amylase concentration even at a low (5%) gelatin concentration. Anti-amylase labeling in ultra-thin sections from Lowicryl K4M low temperature-embedded blocks was higher than in PAA sections, but the results were less consistent and depended to some extent on matrix density. These results, together with the earlier observation that acrylamide completely penetrates intracellular compartments (Slot JW, Geuze HJ: Biol Cell 44:325, 1982), demonstrate that it is possible to measure true intracellular concentrations of soluble proteins in situ using ultra-thin cryosections of PAA-embedded tissue.

scholarly journals Calmodulin immunoelectron microscopy: redistribution during ram spermatogenesis and epididymal maturation. II.

1986 ◽  
Vol 34 (9) ◽  
pp. 1181-1193 ◽  
Author(s):  
S Weinman ◽  
C Ores-Carton ◽  
F Escaig ◽  
J Feinberg ◽  
S Puszkin
Keyword(s):  
Male Gamete ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Epididymal Maturation ◽  
Coarse Fibers ◽  
Preferential Location ◽  
Lowicryl K4m ◽  
Pleiotropic Regulator ◽  
Monospecific Antibodies

Affinity-purified monospecific antibodies and indirect immunogold and immunoferritin labeling on ultra-thin sections of low-temperature Lowicryl K4M-embedded samples were used to study the redistribution of calmodulin in ram spermatids and epididymal spermatozoa at the electron microscopic level. Calmodulin appeared as an integral component of well-defined structures or organelles of these cells. In young spermatids, calmodulin was localized in the nucleus, cytoplasm, and developing acrosome. During spermatogenesis and epididymal maturation, calmodulin left the acrosome to reach the perinuclear substance and finally became concentrated in the post-acrosomal area of the head, although some calmodulin remained associated with the tip of the acrosome. Such a redistribution is consistent with the preferential location of Ca2+ in the post-acrosomal cytoplasm of ejaculated spermatozoa. Calmodulin was also observed in the flagellum associated with the plasma membrane and with the motility apparatus, between coarse fibers and axonemal microtubules. These changes in calmodulin distribution may account for the Ca2+-dependent regulation of spermatogenesis and sperm maturation. Calmodulin therefore appears to be a pleiotropic regulator of male gamete development and functions.

scholarly journals In situ nick translation at the electron microscopic level: a tool for studying the location of DNAse I-sensitive regions within the cell.

1991 ◽  
Vol 39 (6) ◽  
pp. 871-874 ◽  
Author(s):  
M Thiry
Keyword(s):  
Ultrastructural Level ◽  
Dnase I ◽  
Electron Microscopic Level ◽  
Nick Translation ◽  
Dna Polymerase I ◽  
Electron Microscopic ◽  
Thin Sections ◽  
In Situ Nick Translation ◽  
Polymerase I

The in situ nick translation method was adapted to the ultrastructural level, to study the location of DNAse I-sensitive sequences within the cell. Ultra-thin sections of Lowicryl-embedded cells were incubated in a medium containing DNAse I, DNA polymerase I, and all four deoxyribonucleotides, some being biotinylated. The nick-translated sites were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern is closely dependent on the DNAse I concentration in the nick-translation medium. The method reveals with great precision the specific DNAse I-sensitive regions within the nucleus. This technique can be used to discriminate between active and inactive regions of interphase chromatin.

scholarly journals Electron Microscopic Immunogold Localization of Salivary Mucins MG1 and MG2 in Human Submandibular and Sublingual Glands

2003 ◽  
Vol 51 (1) ◽  
pp. 69-79 ◽  
Author(s):  
Marco Piludu ◽  
Sean A. Rayment ◽  
Bing Liu ◽  
Gwynneth D. Offner ◽  
Frank G. Oppenheim ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Expression Patterns ◽  
Cell Types ◽  
Mucous Cells ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Dense Cores ◽  
Duct Cells ◽  
Rabbit Igg ◽  
Lowicryl K4m

The human salivary mucins MG1 and MG2 are well characterized biochemically and functionally. However, there is disagreement regarding their cellular and glandular sources. The aim of this study was to define the localization and distribution of these two mucins in human salivary glands using a postembedding immunogold labeling method. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the submandibular and sublingual glands were intensely reactive with anti-MG1. No reaction was detected in serous cells. With anti-MG2, the granules of both mucous and serous cells showed reactivity. The labeling was variable in both cell types, with mucous cells exhibiting a stronger reaction in some glands and serous cells in others. In serous granules, the electron-lucent regions were more reactive than the dense cores. Intercalated duct cells near the acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, the basal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These results confirm those of earlier studies on MG1 localization in mucous cells and suggest that MG2 is produced by both mucous and serous cells. They also indicate differences in protein expression patterns among salivary serous cells.

scholarly journals Application of lectin--gold complexes for electron microscopic localization of glycoconjugates on thin sections.

1983 ◽  
Vol 31 (8) ◽  
pp. 987-999 ◽  
Author(s):  
J Roth
Keyword(s):  
Endoplasmic Reticulum ◽  
Concanavalin A ◽  
Binding Sites ◽  
Light And Electron Microscopy ◽  
Gold Complex ◽  
Electron Microscopic ◽  
Gold Complexes ◽  
Thin Sections ◽  
Renal Tubular Cells ◽  
Lowicryl K4m

A method is described for the electron microscopic detection of lectin-binding sites in different cellular compartments and extracellular structures that uses thin sections from resin-embedded tissues. Various lectins (Ricinus communis lectin I and II, peanut lectin, Lotus tetragonolobus lectin, Ulex europeus lectin I, Lens culinaris lectin, Helix pomatia lectin, and soybean lectin) were bound to particles of colloidal gold and used for direct staining of thin sections or glycoprotein--gold complexes were prepared and applied in an indirect technique (concanavalin A and horseradish peroxidase--gold complex; wheat germ lectin and ovomucoid--gold complex). The details for preparation of such complexes from 14 nm gold particles are reported. The conditions of tissue processing that gave satisfactory staining results and good fine structure preservation were mild aldehyde fixation without osmification and low temperature embedding with the hydrophilic resin Lowicryl K4M. None of the so-called etching procedures was necessary prior to labeling of Lowicryl K4M thin sections. Examples of the use of this approach for detection of glycoconjugates in the rough endoplasmic reticulum, Golgi apparatus, and mucin of intestinal goblet cells as well as plasma membrane and various intracellular structures of absorptive intestinal and renal tubular cells are shown. A comparison is made with preembedding staining results on Concanavalin A-binding site localization in rat liver which shows that problems of penetration common in such a technique are circumvented by the postembedding approach described here. Concanavalin A-binding sites were not only consistently found in nuclear envelope, rough and smooth endoplasmic reticulum, plasma membranes, and collagen fibers, but also in mitochondria, glycogen, ribosomes, and nucleus. These data and those of a previous investigation (Roth J, Cytochem 31:547, 1983) prove the applicability of this cytochemical technique for postembedding localization of glycoconjugates by light and electron microscopy.

In Situ Hybridization at the Electron Microscopic Level Using a Bromodeoxyuridine Labeled DNA Probe

1993 ◽  
Vol 68 (3) ◽  
pp. 169-174 ◽  
Author(s):  
Chong-Hua Yao ◽  
Sohei Kitazawa ◽  
Takahiro Fujimori ◽  
Sakan Maeda
Keyword(s):  
In Situ Hybridization ◽  
Microscopic Level ◽  
Dna Probe ◽  
Electron Microscopic Level ◽  
Electron Microscopic

scholarly journals Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

1992 ◽  
Vol 40 (11) ◽  
pp. 1647-1657 ◽  
Author(s):  
R W Dirks ◽  
A G Van Dorp ◽  
J Van Minnen ◽  
J A Fransen ◽  
M Van der Ploeg ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Lymnaea Stagnalis ◽  
Pond Snail ◽  
28S Rrna ◽  
Electron Microscopic ◽  
Rna Sequences ◽  
Double Labeling ◽  
Thin Sections ◽  
Cell Hormone

The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea stagnalis was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28S rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable preservation of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphology, but on Lowicryl sections this added fixative resulted in a decrease of label intensity. A variety of probes, including plasmids, PCR products, and oligonucleotides, were used and all provided good results, although the use of oligonucleotides on Lowicryl sections resulted in decreased gold labeling. The gold particles were found mainly associated with rough endoplasmic reticulum (RER) but were also observed in lysosomal structures. Finally, the in situ hybridization method presented in this study proved to be compatible with the immunocytochemical detection of the caudodorsal cell hormone, as demonstrated by double labeling experiments.

scholarly journals Fossil fern rhizomes as a model system for biotic interactions across geologic time: Evidence from Patagonia

2017 ◽  
Author(s):  
Alexander C. Bippus ◽  
Ignacio H. Escapa ◽  
Peter Wilf ◽  
Alexandru M. F. Tomescu
Keyword(s):  
Fossil Record ◽  
Oribatid Mite ◽  
Biotic Interactions ◽  
Terrestrial Ecosystems ◽  
Model System ◽  
Thin Sections ◽  
Geologic Time ◽  
Ancient Ecosystems ◽  
Plant Plant

Background. A wealth of data on the networks of ecological interactions present in the modern biota can be readily obtained, due to the ease of unlimited access to the living organisms that form these networks. In contrast, understanding of such interactions in ecosystems of the geologic past is incomplete. Specifically, in terrestrial ecosystems we know comparatively little about plant biotic interactions besides herbivory, oviposition, galling. Here we describe a tiny in situ fossil community which sheds light on concurrent plant-plant, plant-fungal, and plant-animal interactions. Methods. A single silicified osmundaceous rhizome from a new locality of the early Eocene (ca. 52 Ma) Tufolitas Laguna del Hunco was studied in serial thin sections using light microscopy. The community of organisms colonizing the tissues of the rhizome was characterized by identifying the organisms, as well as mapping and quantifying their distribution. For this, a 200 x 200 µm grid was superimposed onto the rhizome cross section and the colonizers present at each node of the grid were tallied. Results. Preserved in situ, this community offers a rare window onto aspects of ancient ecosystems usually lost to time and taphonomic processes. The community is surprisingly diverse and includes the first fossilized leafy liverworts in South America, also marking the only fossil record of leafy bryophyte epiphytes; several types of fungal hyphae and spores; microsclerotia with probable affinities in several ascomycete families; and oribatid mite coprolites. Discussion. The community associated with the Patagonian rhizome enriches our understanding of plant biotic interactions in the distant past and adds to a growing body of literature, which indicates that osmundaceous rhizomes were important hosts for component communities in ancient ecosystems, just as they are today. Because osmundaceous rhizomes represent an ecological niche that has remained unchanged over time and space, and are abundant in the fossil record, they provide a good paleoecological model system that could be used for exploring plant biotic interactions across geologic time.

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