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 Ultrastructural localization of acidic glycoconjugates with the low iron diamine method.

1982 ◽  
Vol 30 (5) ◽  
pp. 471-476 ◽  
Author(s):  
M Takagi ◽  
R T Parmley ◽  
S S Spicer ◽  
F R Denys ◽  
M E Setser
Keyword(s):  
Bone Marrow ◽  
External Surface ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Electron Microscope Level ◽  
Thin Sections

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.

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 THE ENDOPLASMIC RETICULUM OF GASTRIC PARIETAL CELLS

1961 ◽  
Vol 11 (2) ◽  
pp. 333-347 ◽  
Author(s):  
Susumu Ito
Keyword(s):  
Endoplasmic Reticulum ◽  
Continuous System ◽  
Parietal Cells ◽  
Secretory Cells ◽  
Cell Type ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Oxyntic Cell ◽  
Lower Vertebrates ◽  
Gastric Parietal Cells

An electron microscopic survey has been made of the gastric parietal or oxyntic cell of the human, cat, beaver, dog, hamster, rat, mouse, and bat, and of the corresponding cell type in two species of frog, two species of toad, and the horned lizard. A feature consistently found in the parietal cells of the mammals or their equivalent in the lower vertebrates is the agranular endoplasmic reticulum, which takes the form of branching and anastomosing small tubules approximately 200 to 500 A in diameter, sometimes expanded into flattened cisternae. In mammalian parietal cells this form of the endoplasmic reticulum is found only in limited amounts, but in the corresponding secretory cells of the amphibia and reptilia the tubular agranular reticulum is abundant. It is believed to comprise a more or less continuous system of channels, but owing to their tortuous course only short profiles are seen in thin sections. Immediately subjacent to the plasmalemma at the free surface, the cytoplasm is relatively free of organelles but is occasionally traversed by the agranular reticulum, which appears to be continuous at some points with the cell surface. The possible participation of the agranular endoplasmic reticulum in hydrochloric acid secretion is discussed.

scholarly journals Ultrastructural localization of carbonic anhydrase in the chorioallantoic membrane by immunocytochemistry.

1981 ◽  
Vol 29 (10) ◽  
pp. 1121-1127 ◽  
Author(s):  
R E Anderson ◽  
C V Gay ◽  
H Schraer
Keyword(s):  
Chick Embryo ◽  
Carbonic Anhydrase ◽  
Acid Secretion ◽  
Chorioallantoic Membrane ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Apical Vesicles ◽  
Immuno Cytochemistry ◽  
Egg Shell ◽  
Co2 Release

Carbonic anhydrase was localized in the chick embryo chorionic ectoderm at the ultrastructural level by immuno-cytochemistry. Preembedding staining of whole tissue was performed. The enzyme was present in the cytoplasm, on the membranes of apical vesicles, and on the membranes of microvilli in villus cavity cells, cells that may be involved in acid secretion and subsequent dissolution of the egg shell. In sinus covering cells, the enzyme is solely in the cytoplasm. The location of the enzyme in the thin cytoplasmic arms of the sinus covering cells is consistent with the role in nonrespiratory CO2 release.

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.

scholarly journals Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold.

1985 ◽  
Vol 33 (10) ◽  
pp. 1015-1025 ◽  
Author(s):  
M Castel ◽  
J Morris ◽  
Y Ben-Barak ◽  
R Timberg ◽  
N Sivan ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Secretory Granules ◽  
Picric Acid ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Neurosecretory Granules ◽  
Sodium Metaperiodate ◽  
Background Staining ◽  
Axonal Varicosity

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.

scholarly journals High-resolution cytochemistry of neuraminic and hexuronic acid-containing macromolecules applying the enzyme-gold approach.

1988 ◽  
Vol 36 (8) ◽  
pp. 1005-1014 ◽  
Author(s):  
I Londoño ◽  
M Bendayan
Keyword(s):  
Golgi Apparatus ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Goblet Cells ◽  
Ultrastructural Level ◽  
Gold Complex ◽  
Thin Sections ◽  
Gold Particles ◽  
Tissue Sections ◽  
Apical Side

We localized acidic glycoconjugates at the ultrastructural level by applying the enzyme-gold approach. Neuraminidase and hyaluronidase were adsorbed to colloidal gold particles and applied to tissue sections under optimal conditions for their enzymatic activity. Neuraminidase-gold labeling was distributed over the Golgi apparatus and associated secretory granules in exocrine pancreatic cells and duodenal goblet cells. Mitochondria were labeled over inner membranes. Labeling was also found over the dispersed chromatin in the nucleus. Plasma membranes, particularly the apical side, were labeled by gold particles. On the other hand, incubation of tissue sections with the hyaluronidase-gold complex resulted in intense labeling of the rER membranes, the plasma membrane, and the dense chromatin in the nucleus. Labeling was also found over the Golgi apparatus and associated secretory granules, but only in duodenal goblet cells. Specificity of the results was confirmed by various control experiments performed, indicating that the enzyme-gold technique is useful for detecting linked-sugar residues on tissue thin sections. Labelings found over intra- and extracellular compartments in the present work are discussed in light of previous biochemical indications as well as of other histochemical detections of these glycoconjugates.

scholarly journals Ia antigens in plastic-embedded tissues: a post-embedding immunohistochemical study.

1986 ◽  
Vol 34 (6) ◽  
pp. 827-831 ◽  
Author(s):  
W Hermanns ◽  
F Colbatzky ◽  
A Günther ◽  
B Steiniger
Keyword(s):  
Rat Tissues ◽  
Thin Sections ◽  
Tissue Sections ◽  
Histocompatibility Complex ◽  
Embedding Technique ◽  
Ia Antigens ◽  
Distinct Cell ◽  
Plastic Embedding ◽  
Indirect Immunoperoxidase ◽  
Cryostat Sections

The aim of the present study was to establish a plastic embedding technique that makes possible the immunohistochemical demonstration of class II major histocompatibility complex (MHC) antigens (Ia antigens) in undecalcified joint tissues. Therefore a series of fixatives and dehydrating agents was tested for saving Ia immunoreactivity by post-embedding immunostaining of thin sections (2 microns) of rat tissues that had been embedded in glycol methacrylate (GMA), and by comparing with cryostat sections. An indirect immunoperoxidase and the avidin-biotin complex (ABC) technique were used. Combined with fixation by 4% formaldehyde, dehydration with GMA was found to give the best preservation of Ia antigenicity, followed by dehydration with ethylene glycol. The thinness of tissue sections facilitated the association of Ia antigens with different subcellular compartments in distinct cell populations. These various patterns are described.

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