scholarly journals Ultrastructural immunocytochemical localization of 5-hydroxytryptamine in gastric enterochromaffin cells.

1984 ◽  
Vol 32 (6) ◽  
pp. 661-666 ◽  
Author(s):  
R D Dey ◽  
J Hoffpauir
Keyword(s):  
Electron Microscopy ◽  
Potassium Dichromate ◽  
Picric Acid ◽  
Ultrastructural Localization ◽  
Immunocytochemical Localization ◽  
Thin Sections ◽  
Glass Slides ◽  
Ec Cells ◽  
Block Face ◽  
Immunocytochemical Method

Enterochromaffin (EC) cells in the gastrointestinal tract are known to contain 5-hydroxytryptamine (5HT). The probable ultrastructural localization of 5HT in the dense core vesicles ( DCVs ) of EC cells is based on the use of histochemical techniques, such as argentaffinity and the potassium dichromate reaction. In the present paper we describe an immunocytochemical method for specifically localizing 5HT in EC cells by electron microscopy. Pieces of mucosa from the pyloric region of the rabbit stomach were prepared for electron microscopy by fixation in 0.5% glutaraldehyde-picric acid-formaldehyde without osmication , and then embedded in LX-112. Thick sections (1 micron) were mounted on glass slides and processed for the fluorescence immunocytochemical localization of 5HT. Thin sections (60-90 nm) were mounted on formvar-coated slot grids and processed for the ultrastructural immunocytochemical localization of 5HT. Both the thick and thin sections were processed by an identical procedure, beginning with a 30-min incubation in anti-5HT antiserum diluted 1:1400, followed by an IgG-FITC-gold-labeled second antibody. Fluorescent EC cells were consistently observed in the thick sections of gastric mucosa. By carefully trimming and sectioning the adjacent block face, the identical EC cell could be identified by electron microscopy. A quantitative analysis revealed the number of gold particles in EC cells to be significantly greater over the cores of DCVs than over the non-core cytoplasm or over the nucleus. Absorption of the primary antiserum with 5HT abolished all labeling, while absorption with a 5HT precursor, 5-hydroxytryptophan, did not significantly reduce core labeling. Non-EC epithelial cells were not labeled. These results demonstrate that immunoreactive 5HT in EC cells is stored in the cores of DCVs .

scholarly journals An improved immunocytochemical method for subcellular localization of serotonin in rat enterochromaffin cells.

1987 ◽  
Vol 35 (3) ◽  
pp. 319-326 ◽  
Author(s):  
O Nilsson ◽  
A Dahlström ◽  
M Geffard ◽  
H Ahlman ◽  
L E Ericson
Keyword(s):  
Osmium Tetroxide ◽  
Secretory Granules ◽  
Protein A ◽  
Ultrastructural Level ◽  
Proximal Colon ◽  
Intracellular Distribution ◽  
Thin Sections ◽  
Ec Cells ◽  
Immunocytochemical Method ◽  
A Minor

Serotonin-like immunoreactivity (5-HT-LI) has been localized at the ultrastructural level in enterochromaffin (EC) cells of rat gastrointestinal tract. Ultra-thin sections of tissues embedded in epoxy resin were incubated with 5-HT antisera and antibody binding sites were visualized with protein A-gold. Three different antisera were compared and were shown to require different fixation regimens for optimal preservation of 5-HT-LI. For one antiserum, tissues fixed in glutaraldehyde and osmium tetroxide could be used to demonstrate 5-HT-LI in EC cells. Immunocytochemical localization of 5-HT can thus be performed with good ultrastructural preservation of tissues. Quantitative evaluation of the intracellular distribution of 5-HT-LI was performed on EC cells from antrum, duodenum, and proximal colon, fixed in glutaraldehyde only. In all three locations, the majority of the gold particles (90%) in EC cells were localized over the dense core of the secretory granules, while a minor fraction (10%) were localized in parts of the cytoplasm devoid of granules. In EC cells fixed in glutaraldehyde and post-fixed in osmium tetroxide, 5-HT-LI was reduced by about 85%, although intracellular distribution was essentially the same as in cells fixed in glutaraldehyde alone. The results indicate that 5-HT in EC cells is stored mainly in secretory granules, with a small fraction of 5-HT being localized outside the granules.

scholarly journals Identification of the APUD endocrine cells of rat fundic mucosa by means of combined amine fluorescence and electron microscopy.

1984 ◽  
Vol 32 (1) ◽  
pp. 67-75 ◽  
Author(s):  
W Rubin ◽  
B Schwartz
Keyword(s):  
Electron Microscopy ◽  
Endocrine Cells ◽  
Thin Sections ◽  
Fundic Mucosa ◽  
Pyloric Mucosa ◽  
Fluorescence And Electron Microscopy ◽  
Freeze Dried ◽  
Rat Gastric Mucosa ◽  
Ec Cells ◽  
D Cells

Rat fundic mucosa contains numerous APUD endocrine cells that can take up 5-hydroxytryptophan (5-HTP) and decarboxylate it to 5-hydroxytryptamine (serotonin), detectable by its formaldehyde-induced yellow fluorescence. To identify these cells by electron microscopy, pieces of rat gastric mucosa were incubated with DL-5-HTP. Some specimens were fixed in 4% formaldehyde-0.5% glutaraldehyde, while others were frozen, freeze-dried, and exposed to paraformaldehyde vapors. Thick sections of the Epon-embedded specimens were examined and photographed by fluorescence microscopy, and fluorescing and nonfluorescing cells were identified by electron microscopy in serial thin sections. Control specimens, not incubated with 5-HTP, revealed fluorescence of only the mast cells and EC cells, which were abundant in the pylorus, but rare in fundic mucosa. Specimens incubated with 5-HTP also exhibited numerous yellow fluorescent endocrine cells in fundic mucosa, which cells were found to be the ECL cells; the A-like cells did not fluoresce. In pyloric mucosa, many G and D (D1) cells also exhibited weak or moderate fluorescence after 5-HTP incubation. Thus, this study supports the contention of previous radioautographic studies that the ECL, but not the A-like, cells are the APUD endocrine cells of rat fundic mucosa, that G and D cells also possess some APUD activity, and that EC cells represent the enterochromaffin cells, which normally synthesize and store demonstrable quantities of serotonin.

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.

Organization of the cytoskeleton during cellularization in Drosophila melanogaster embryos: Confocal laser scanning and electron microscopy

1990 ◽  
Vol 48 (3) ◽  
pp. 462-463
Author(s):  
Kazuo Katoh ◽  
Harunori Ishikawa
Keyword(s):  
Electron Microscopy ◽  
Drosophila Melanogaster ◽  
Phosphate Buffer ◽  
Laser Scanning ◽  
Picric Acid ◽  
Egg Laying ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Thin Sections

The three-dimensional organization of cytoskeletal components in the early Drosophila melanogaster embryos during cellularization was examined by confocal laser scanning microscopy of the whole embryos and thin section electron microscopy.For confocal microscopy, Drosophila embryos at 2-3 hr after egg-laying were dechorionated and fixed with 8% paraformaldehyde-1% picric acid in 0.1M phosphate buffer (pH 7.2). Embryos were first blocked with normal goat serum, incubated with monoclonal antibody raised against Drosophila embryo α-tubulin for 4 hr, and then were incubated with FITC-conjugated anti-mouse IgG for 2 hr. Some embryos were stained with rhodamine-labeled phailoidin for F-actin visualization. After staining, the whole embryos were mounted on slide glass with an appropriate spacer and examined under the confocal microscope (Bio-Rad, Lasersharp MRC-500). For electron microscopy, dechorionated embryos were fixed with 1/2 Karnovsky's fixative followed by OsO4 fixation. To better preserve actin filaments, embryos were fixed with the same 1/2 Karnovsky's fixative containing 10 μM phailoidin and 0.1% saponin in 0.1M phosphate buffer, pH 7.2. Such fixed embryos were dehydrated and then embedded in Epoxy resin. Thin sections were cut and examined under a Hitachi H-800 type electron microscope.

scholarly journals Carbonic anhydrase, ultrastructural localization in the mouse gastric mucosa and improvements in the technique.

1980 ◽  
Vol 28 (6) ◽  
pp. 511-525 ◽  
Author(s):  
N Sugai ◽  
S Ito
Keyword(s):  
Electron Microscopy ◽  
Gastric Mucosa ◽  
Carbonic Anhydrase ◽  
Picric Acid ◽  
Ultrastructural Localization ◽  
Carbonic Anhydrase Activity ◽  
Cobalt Sulfide ◽  
Ultrastructural Studies ◽  
Mucosal Cells ◽  
Erythrocyte Carbonic Anhydrase

The ultrastructural localization of carbonic anhydrase activity in mouse gastric mucosal cells as revealed by the cobalt bicarbonate histochemical method of Hansson has been made. In addition the effects of fixatives used for ultrastructural studies have been evaluated for reduction of carbonic anhydrase activity; exogenous erythrocyte carbonic anhydrase has been localized in tissues; acetazolamide and potassium cyanate inhibition of activity demonstrated; and an improved method for the osmication of reacted tissues for electron microscopy has been developed. The results indicate that the glutaraldehyde, formaldehyde, picric acid fixative, which retains about 5% of the original carbonic anhydrase activity, is distinctly better for histochemical studies than formaldehyde fixation, which retains about 32% activity. Acetazolamide at 10(-5) M consistently inhibits histochemical reaction, as does 20 mM KCNO, in the incubation medium. Exogenous carbonic anhydrase is readily visualized by the histochemical technique. Electron microscopy of gastric mucosa reacted for carbonic anhydrase activity indicates the focal deposition of the cobalt sulfide reaction product in the cores of microvilli lining the intracellular canaliculi, in the basal and lateral cell folds of parietal cells, and in the microvilli as well as the cytoplasm between mucous granules in the surface mucous cells. In addition, some reaction product was found in the mitochondrial cristae and in some nuclei and intercellular spaces.

Ultrastructural localization of carbonic anhydrase in mouse gastric parietal cells

1978 ◽  
Vol 36 (2) ◽  
pp. 532-533
Author(s):  
N. Sugai ◽  
S. Ito
Keyword(s):  
Carbonic Anhydrase ◽  
Picric Acid ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Parietal Cells ◽  
Thin Sections ◽  
Tissue Sections ◽  
Gastric Parietal Cells ◽  
Cryostat Sections ◽  
Precise Distribution

The histochemical localization of carbonic anhydrase in parietal cells has been described by a number of investigators and its presence in these cells at the ultrastructural level has been also reported. However the precise distribution of this enzyme is not clear and there are some questions regarding the validity of the histochemical reaction. In the present study, various modifications of the technique were explored and it was found that tissues fixed in buffered formaldehyde, glutaraldehyde with trinitrocresol or picric acid retained good reaction product localization of this enzyme. Cryostat sections of the fixed tissues were treated with solutions recommended by Hannson. Incubation times that were most favorable 5 to 10 min for light microscopy and 8 to 15 min for electron microscopy. For ultrastructural observations of thin sections, it was found to be important that the reacted tissue sections were post osmicated with 1% osmium tetroxide in 1. 5% potassium ferrocyanide or with aqueous 1% 0s04 for only 3 to 5 min.

scholarly journals Antigen unmasking for immunoelectron microscopy: labeling is improved by treating with sodium ethoxide or sodium metaperiodate, then heating on retrieval medium.

1995 ◽  
Vol 43 (2) ◽  
pp. 115-123 ◽  
Author(s):  
J W Stirling ◽  
P S Graff
Keyword(s):  
Electron Microscopy ◽  
Immunoglobulin G ◽  
Biopsy Specimen ◽  
Sodium Ethoxide ◽  
Ultrastructural Localization ◽  
Citrate Buffer ◽  
Sodium Metaperiodate ◽  
Thin Sections ◽  
High Probe Density ◽  
Corneal Biopsy

To optimize the ultrastructural localization of immunoglobulin G in corneal crystalloid deposits, we compared a range of antigen unmasking techniques. A human corneal biopsy specimen was fixed in formalin, post-osmicated, and embedded in epoxy resin for electron microscopy. Thin sections were immunogold-labeled for IgG after treatment with sodium ethoxide or sodium metaperiodate. Sections were also treated by heating them at 95 degrees C while they floated on water, 0.01 M citrate buffer (pH 6.0), or sodium metaperiodate. The treatments were applied separately and combined. After labeling, crystalloids in untreated sections had a probe density of 5 particles/microns2. Crystalloids in sections treated only with sodium ethoxide or sodium metaperiodate had probe densities of 15-20 particles/microns2. Sodium ethoxide combined with heating on water, or citrate buffer, gave probe densities of 140-160 particles/microns2. Sodium metaperiodate combined with heating on citrate buffer gave the highest probe density (195 particles/microns2). Although sodium ethoxide coupled with heating increased probe density, the ethoxide etched the sections and caused unacceptable damage. Treatment with sodium metaperiodate followed by heating on citrate buffer is recommended for antigen unmasking. This combination gave a high probe density and sections remained intact, with good ultrastructural detail.

scholarly journals Immunocytochemical localization of prorenin, renin, and cathepsins B, H, and L in juxtaglomerular cells of rat kidney.

1989 ◽  
Vol 37 (11) ◽  
pp. 1689-1697 ◽  
Author(s):  
H Matsuba ◽  
T Watanabe ◽  
M Watanabe ◽  
Y Ishii ◽  
S Waguri ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cathepsin B ◽  
Rat Kidney ◽  
Renal Tissue ◽  
Juxtaglomerular Cells ◽  
Perinuclear Region ◽  
Immunocytochemical Localization ◽  
Double Immunostaining ◽  
Thin Sections ◽  
Anti Body

To examine the correlation of localization of prorenin, renin, and cathepsins B, H, and L, immunocytochemistry was applied to rat renal tissue, using a sequence-specific anti-body (anti-prorenin) that recognizes the COOH terminus of the rat renin prosegment. In serial semi-thin sections, immunodeposits for prorenin, renin, and cathepsins B, H, and L were localized in the same juxtaglomerular (JG) cells. Immunodeposits for renin were detected throughout the cytoplasm of the cells, whereas those for prorenin were detected in the perinuclear region. Immunoreactivity for cathepsin B was stronger than that for cathepsins H and L. By electron microscopy, prorenin was localized in small (immature) granules but not in large mature granules, whereas renin was localized mainly in mature granules. In serial thin sections, prorenin, renin, and cathepsin B were colocalized in the same immature granules containing heterogeneously dense material (intermediate granules). By double immunostaining, co-localization of renin with cathepsins B, H, or L was demonstrated in mature granules. The results suggest the possibility that processing of prorenin to renin occurs in immature granules of rat JG cells, and cathepsin B detected in JG cells may be a major candidate for the maturation of renin.

Ultrastructural localization of nuclear antigens during interphase in mouse 3T3 fibroblasts

1989 ◽  
Vol 67 (9) ◽  
pp. 563-574 ◽  
Author(s):  
Nathalie Chaly ◽  
Gerald St. Aubin ◽  
David L. Brown
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Ultrastructural Localization ◽  
Nuclear Pores ◽  
Thin Sections ◽  
Nuclear Ultrastructure ◽  
3T3 Fibroblasts ◽  
Nuclear Antigens ◽  
Internal Component ◽  
Immunofluorescence Labelling

Thin sections of mouse 3T3 fibroblast nuclei labelled by immunoperoxidase with anti-nuclear antibodies I1, PI1, PI2, anti-peripherin, -lamin, and -centromere have been examined in the electron microscope. Staining was compared with the corresponding immunofluorescence labelling patterns, and was correlated with nuclear ultrastructure in conventionally fixed and uranyl-lead stained samples and in unlabelled immunoperoxidase controls. Peripherin was detected at the nuclear rim in a band broader and more irregular than the lamins/lamina. The peripheral components of PI1 and PI2 appear to be localized at nuclear pores and the nuclear envelope, respectively. The internal component of PI1 staining consisted of irregular patches and strands in the nucleoplasm, closely resembling snRNP staining as reported by others. Internal PI2 labelling consisted of spots distributed apparently at random in interchromatinic regions. The spots resembled labelling by antibody I1, but were fewer and more irregular in size. Neither the PI2 nor the I1 spots were centromere associated, nor could they be correlated with specific interchromatinic structures in conventional preparations and in unlabelled controls. The results support the hypothesis that the nucleus is segregated into function-specific domains, distinguished by morphology and (or) composition from surrounding regions of the nucleus.Key words: nuclear matrix, peripherin, immunoperoxidase, electron microscopy, nuclear antigens.

Two- or three-way observations of the identical cell elements within the same sections by LM, TEM and/or SEM

1978 ◽  
Vol 36 (2) ◽  
pp. 82-83 ◽  
Author(s):  
Nakazo Watari ◽  
Yasuaki Hotta ◽  
Yoshio Mabuchi
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Fine Structure ◽  
Light Microscopy ◽  
Thin Sections ◽  
Transmission Electron ◽  
Identical Cell ◽  
Electron Microscopes ◽  
Scanning Electron

It is very useful if we can observe the identical cell elements within the same sections by light microscopy (LM), transmission electron microscopy (TEM) and/or scanning electron microscopy (SEM) sequentially, because, the cell fine structure can not be indicated by LM, while the color is; on the other hand, the cell fine structure can be very easily observed by EM, although its color properties may not. However, there is one problem in that LM requires thick sections of over 1 μm, while EM needs very thin sections of under 100 nm. Recently, we have developed a new method to observe the same cell elements within the same plastic sections using both light and transmission (conventional or high-voltage) electron microscopes.In this paper, we have developed two new observation methods for the identical cell elements within the same sections, both plastic-embedded and paraffin-embedded, using light microscopy, transmission electron microscopy and/or scanning electron microscopy (Fig. 1).

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