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 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 Immunoelectron microscopic localization of calmodulin and phospholipase A2 in spermatozoa. I.

1986 ◽  
Vol 34 (9) ◽  
pp. 1171-1179 ◽  
Author(s):  
S Weinman ◽  
C Ores-Carton ◽  
D Rainteau ◽  
S Puszkin
Keyword(s):  
Plasma Membrane ◽  
Phospholipase A2 ◽  
Male Gamete ◽  
Fibrous Sheath ◽  
Thin Sections ◽  
Calmodulin Binding ◽  
Sperm Mobility ◽  
Coarse Fibers ◽  
Lowicryl K4m ◽  
Phospholipase A2 Activity

Using the Lowicryl K4M embedding technique, together with indirect immunoferritin or immunogold labeling on ultra-thin sections, tubulin, calmodulin and phospholipase A2 were distinctly localized in ejaculated bull spermatozoa. Calmodulin was concentrated on the plasma membrane, nucleus, post-acrosomal substance, and, in lesser amounts, between coarse fibers and axonemal microtubules of the flagellum. Phospholipase A2 was distributed evenly along the plasma membrane, nucleus, acrosome, post-acrosomal substance, and in the flagellum, on mitochondria, fibrous sheath, coarse fibers, between coarse fibers and axonemal microtubules. Antibodies to tubulin labeled only axonemal microtubules, including the central pair of microtubules. Patterns of tubulin labeling were identical when ferritin granule- or gold particle-conjugated antibodies were tested. In agreement with our previous biochemical studies demonstrating calmodulin binding to phospholipase A2, concomitant with enhancement of phospholipase A2 activity (Arch Biochem Biophys 241:413, 1985), the overlapping distribution of calmodulin and phospholipase A2 in several parts of the sperm suggests that these proteins may play a concerted role in male gamete function in preparation for or during fertilization. The distinct distribution of tubulin along flagellum microtubules indicates their special function in sperm mobility.

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.

scholarly journals Immunocytochemical demonstration of ecto-galactosyltransferase in absorptive intestinal cells.

1985 ◽  
Vol 100 (1) ◽  
pp. 118-125 ◽  
Author(s):  
J Roth ◽  
M J Lentze ◽  
E G Berger
Keyword(s):  
Plasma Membrane ◽  
Protein A ◽  
Thin Sections ◽  
Golgi Membranes ◽  
Basal Plasma ◽  
Basolateral Plasma Membrane ◽  
Junctional Complexes ◽  
Lowicryl K4m ◽  
Monospecific Antibodies ◽  
Absorptive Enterocytes

Galactosyltransferase immunoreactive sites were localized in human duodenal enterocytes by the protein A-gold technique on thin sections from low temperature Lowicryl K4M embedded biopsy specimens. Antigenic sites detected with affinity-purified, monospecific antibodies were found at the plasma membrane of absorptive enterocytes with the most intense labeling appearing along the brush border membrane. The lateral plasma membrane exhibited a lower degree of labeling at the level of the junctional complexes but the membrane interdigitations were intensely labeled. The labeling intensity decreased progressively towards the basal part of the enterocytes and reached the lowest degree along the basal plasma membrane. Quantitative evaluation of the distribution of gold-particle label proved its preferential orientation to the outer surface of the plasma membrane. In addition to this membrane-associated labeling, the glycocalyx extending from the microvillus tips was heavily labeled. Occasionally, cells without plasma membrane labeling were found adjacent to positive cells. The demonstration of ecto-galactosyltransferase on membranes other than Golgi membranes precludes its general use as a marker for Golgi membrane fractions. The possible function of galactosyltransferase on a luminal plasma membrane is unclear at present, but a role in adhesion appears possible on the basolateral plasma membrane.

Electron microscopic analysis of objects in light microscopic sections

1978 ◽  
Vol 36 (2) ◽  
pp. 80-81
Author(s):  
Arvid B. Maunsbach
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Microscopic Analysis ◽  
Electron Microscopic Level ◽  
Semithin Section ◽  
Cover Glass ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Glass Slides ◽  
Ultrathin Sections

Structural studies in experimental biology or in pathology are frequently extended from the light to the electron microscopic level. This is often done by cutting both semithin (about 1 μm) and thin sections from the same tissue block after embedding for electron microscopy. However, in many studies it would be of great value to analyse the same structure both by light and electron microscopy, i.e. to be able to study by electron microscopy an object which is first detected by light microscopy in a semithin section. To achieve this, a method has been developed by which ultrathin sections are cut directly from the semithin section containing the object of interest.Semithin sections, about 1 μ in thickness, are cut from Epon or Vestopal embedded tissue. The sections are placed on ordinary glass slides and stained with toluidine blue. The sections are studied in the light microscope without a cover glass or mounted in water.

Correlative immunolocalization with high-resolution light microscopy and Electron Microscopy using London Resin Gold embedded tissue

1995 ◽  
Vol 53 ◽  
pp. 702-703
Author(s):  
M. L. Grove ◽  
B. A. Evans ◽  
D. N. Misra ◽  
J. Zhao ◽  
D. H. Alpers ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Polyclonal Antibodies ◽  
Intracellular Localization ◽  
Intrinsic Factor ◽  
Gastric Epithelium ◽  
Electron Microscopic Level ◽  
Immunoperoxidase Staining ◽  
Rat Tissues ◽  
Electron Microscopic ◽  
Thin Sections

Immunoelectron microscopy specimens are often embedded in hydrophilic resins, like London Resin Gold(LRG), which permit antibody staining without etching (or deplasticizing) the sections. This characteristic of hydrophilic resins also allows for immunohistochemistry at the light level on semi thin sections (0.5μm - 1μm). The ability to do immunohistochemistry at both the light and electron microscopic level on the same tissue block allows focused ultrastructural study. Immunohistochemistry on semi-thin sections displays cellular localization of macromolecules, permitting more specificity in the selection of areas for studying intracellular localization ultrastructurally. We have developed a method for immunoperoxidase staining of LRG embedded tissues, utilizing anti-human polyclonal antibodies directed against intrinsic factor (Fig. 1). Intrinsic factor (IF), a cobalamin binding protein, is known to be produced in the stomach, pancreas and salivary glands of most mammals. We are interested in distribution of IF in gastric epithelium, small intestine (ileum) and supporting tissues in both gastrointestinal tract sites. Previously, we have described the cellular localization of IF in human and rat tissues.

scholarly journals Use of anti-horseradish peroxidase antibody-gold complex in the ABC technique.

1991 ◽  
Vol 39 (6) ◽  
pp. 863-869 ◽  
Author(s):  
B Gee ◽  
M J Warhol ◽  
J Roth
Keyword(s):  
Horseradish Peroxidase ◽  
Polyclonal Antibodies ◽  
Light And Electron Microscopy ◽  
Gold Complex ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Endogenous Peroxidase ◽  
Pre Treatment ◽  
Lowicryl K4m ◽  
Antigen Antibody

We report a modification of the avidin-biotin-peroxidase complex (ABC) technique for the light and electron microscopic detection of antigens in tissue sections. An immunological approach was used instead of the DAB reaction to reveal ABC bound to antigen-antibody complexes. Affinity-purified polyclonal antibodies against horseradish peroxidase were complexed to particles of colloidal gold and applied for reaction with the horseradish peroxidase molecules of the ABC. For light microscopic immunolabeling, the signal produced by the anti-horseradish peroxidase antibody-gold complex required silver intensification. The ABC immunogold reaction as compared with the standard ABC technique, in particular with silver intensification of the DAB reaction product, provided superior resolution in paraffin sections. Furthermore, section pre-treatment to block endogenous peroxidase activity could be omitted and no potentially hazardous substrate was used. The ABC immunogold reaction was successfully applied for electron microscopic immunolabeling on Lowicryl K4M thin sections. We propose that the ABC immunogold reaction is a useful alternative to the standard ABC technique and can be equally well applied to light and electron microscopy.

scholarly journals Light and electron microscopic demonstration of sialic acid residues with the lectin from Limax flavus: a cytochemical affinity technique with the use of fetuin-gold complexes.

1984 ◽  
Vol 32 (11) ◽  
pp. 1167-1176 ◽  
Author(s):  
J Roth ◽  
J M Lucocq ◽  
P M Charest
Keyword(s):  
Electron Microscopy ◽  
Sialic Acid ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
Gold Complexes ◽  
Thin Sections ◽  
Tissue Sections ◽  
Lowicryl K4m ◽  
Hydrolysis Of ◽  
Electron Microscopic Demonstration

The development of a cytochemical affinity technique for the demonstration of sialic acid residues by light and electron microscopy is reported. The lectin from the slug Limax flavus, with its narrow specificity for N-acetyl- and N-glycolylneuraminic acid, was applied to tissue sections. Subsequently fetuin-gold complexes were used to visualize the tissue-bound lectin. Different cytochemical controls, including sugar inhibition tests, neuraminidase digestion, the use of fetuin-gold complexes alone, or acid hydrolysis of sections, proved the specificity of the technique. Postembedding staining was performed on frozen, paraffin, or semithin resin sections for light microscopy and on thin sections from low temperature Lowicryl K4M-embedded material for electron microscopy. The distribution of sialic acid residues in rat pancreas, liver, and colonic mucosa was investigated.

scholarly journals A simple and reliable method for correlative light and electron microscopic studies.

1993 ◽  
Vol 41 (5) ◽  
pp. 769-772 ◽  
Author(s):  
J DeFelipe ◽  
A Fairén
Keyword(s):  
Golgi Method ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Simple Method ◽  
Thin Sections ◽  
Axonal Arborization ◽  
Accurate Localization ◽  
Mouse Cerebral Cortex ◽  
Microscopic Studies ◽  
Flat Embedding

We describe in detail a simple method for flat-embedding that can be subsequently used in correlative light and electron microscopic studies. The method can be applied to any material suitable for electron microscopy and is especially useful for study of the synaptology and ultrastructural characteristics of immunocytochemically or morphologically identified neurons or their processes. We present here an example to show how accurately one can delineate the fine details of a complex axonal arborization impregnated with the Golgi method in the mouse cerebral cortex. Golgi-impregnated sections to be studied at the electron microscopic level are osmicated, dehydrated, infiltrated with Araldite resin, flat-embedded, and identified cells or processes photographed. Serial semi-thin sections (1-2 microns thick) are then cut with an ultramicrotome, examined with the light microscope, and the elements rephotographed. Selected semi-thin sections are then resectioned on the ultramicrotome at 60-70 nm and examined electron microscopically. This method allows the systematic and accurate localization of stained cells and processes throughout the successive steps of the procedure.

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