scholarly journals ULTRASTRUCTURAL LOCALIZATION OF CONTRACTILE PROTEIN (THROMBOSTHENIN) IN HUMAN PLATELETS USING AN UNLABELED ANTIBODY-PEROXIDASE STAINING TECHNIQUE

1971 ◽  
Vol 19 (9) ◽  
pp. 540-550 ◽  
Author(s):  
FRANCOIS M. BOOYSE ◽  
DOROTHEA ZSCHOCKE ◽  
MAX E. RAFELSON ◽  
LUDWIG A. STERNBERGER
Keyword(s):  
Horseradish Peroxidase ◽  
Staining Technique ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Contractile Protein ◽  
Intact Cells ◽  
Proteinaceous Material ◽  
Antibody Staining ◽  
Ultrathin Sections ◽  
Unlabeled Antibody

Soluble horseradish peroxidase-antihorseradish peroxidase (rabbit) complex was used to localize the actomyosin-like contractile protein, thrombosthenin, in both intact human platelets (Epon-embedded) and ultrathin sections (methacrylate-embedded). Antibody staining of ultrathin sections showed the presence of membrane-associated and cytoplasmic thrombosthenin. Antibody staining of intact cells showed that membrane-associated thrombosthenin was localized, at least in part, in the exterior, "fluffy" coat of the platelet. Even after prolonged fixation and/or incubation with the various antisera, we were unable to demonstrate antibody penetration of intact, fixed platelets. Brief treatment with Pronase removed the surface-localized proteinaceous material and completely abolished all antibody staining.

scholarly journals Ultrastructural immunohistochemical localization of anti-invasion factor (AIF) in bovine cartilage matrix.

1982 ◽  
Vol 30 (6) ◽  
pp. 524-531 ◽  
Author(s):  
F H Wezeman ◽  
G V Childs
Keyword(s):  
Staining Technique ◽  
Ultrastructural Localization ◽  
Immunohistochemical Localization ◽  
Cartilage Matrix ◽  
Fixed Tissue ◽  
Antibody Complex ◽  
Protein Components ◽  
Antigen Antibody ◽  
Unlabeled Antibody ◽  
Bovine Cartilage

Rabbit antibodies prepared against bovine cartilage anti-invasion factor (AIF) were tested for their affinity toward antigenic sites in glutaraldehyde-fixed bovine hyaline cartilage matrix. Ultrastructural localization of the antigen-antibody complex was accomplished by the unlabeled antibody peroxidase-antiperoxidase staining technique. Unextracted and salt-extracted (1 M NaCl or 3 M GuHCl) cartilage slices were incubated with anti-AIF antibodies at a working dilution of 1:20,000. Staining occurred in unextracted matrix distributed throughout the tissue, but with regional variation in the lacunar matrix. Significantly less stain was noted in extracted tissues. The results suggest that at least certain protein components in AIF are morphologically associated with matrix complexes in aldehyde-fixed tissue.

Ultrastructural localization of neuropeptides in the rat brain

1978 ◽  
Vol 36 (2) ◽  
pp. 612-613
Author(s):  
F.W. Van Leeuwen
Keyword(s):  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Serial Sections ◽  
Microscopical Level ◽  
Electron Microscopical Level ◽  
Enzyme Method ◽  
Perfusion Fixation ◽  
Electron Microscopical ◽  
Unlabeled Antibody ◽  
Peroxidase Conjugate

In order to obtain specific and optimal ultrastructural localization of vasopressin and oxytocin in the hypothalamo-neurohypophyseal system of the rat, 2 staining procedures and several tissue treatments were evaluated using neurohypophyseal tissue. It appeared from these studies that post-embedding staining with the unlabeled antibody enzyme method developed by Sternberger allows greater dilution of the first antibody (anti-vasopressin, 1:4800) than the indirect procedure (1:320) using a peroxidase conjugate as second antibody. Immersion fixation with 4% formalin during 24 hours gave better results (general ultrastructure, immunoreactivity) than those obtained by perfusion fixation with 2.5% glutaraldehyde-1% paraformaldehyde or freeze substitution.Since no reliable specificity tests were performed at the electron microscopical level, tests were developed for antibodies against both vasopressin and oxytocin. For anti-vasopressin plasma neural lobes of homozygous Brattleboro rats, that are lacking vasopressin by a genet- ical defect, were used. For antibodies against both hormones serial sections were used that were alternately incubated with the antibodies.

Ferritin Labeled Antibody Staining of Thin Sections

1969 ◽  
Vol 27 ◽  
pp. 420-421
Author(s):  
J. D. McLean ◽  
S. J. Singer
Keyword(s):  
Biological Material ◽  
Water Soluble ◽  
Thin Sections ◽  
Antibody Staining ◽  
Thin Sectioning ◽  
Ultrathin Sections

The successful application of ferritin labeled antibodies (F-A) to ultrathin sections of biological material has been hampered by two main difficulties. Firstly the normally used procedures for the preparation of material for thin sectioning often result in a loss of antigenicity. Secondly the polymers employed for embedding may non-specifically absorb the F-A. Our earlier use of cross-linked polyampholytes as embedding media partially overcame these problems. However the water-soluble monomers used for this method still extract many lipids from the material.

Comparative strengths and weaknesses of peroxidase and colloidal gold in pre- and post-embedding immunolabeling techniques

1987 ◽  
Vol 45 ◽  
pp. 1002-1005
Author(s):  
D. R. Abrahamson ◽  
P. L. St.John ◽  
E. W. Perry
Keyword(s):  
Electron Microscopy ◽  
Horseradish Peroxidase ◽  
Light Microscopy ◽  
Colloidal Gold ◽  
Light Microscope ◽  
Ultrastructural Localization ◽  
The Other ◽  
Quantitative Studies ◽  
Dense Suspension ◽  
Antigen Localization

Antibodies coupled to tracers for electron microscopy have been instrumental in the ultrastructural localization of antigens within cells and tissues. Among the most popular tracers are horseradish peroxidase (HRP), an enzyme that yields an osmiophilic reaction product, and colloidal gold, an electron dense suspension of particles. Some advantages of IgG-HRP conjugates are that they are readily synthesized, relatively small, and the immunolabeling obtained in a given experiment can be evaluated in the light microscope. In contrast, colloidal gold conjugates are available in different size ranges and multiple labeling as well as quantitative studies can therefore be undertaken through particle counting. On the other hand, gold conjugates are generally larger than those of HRP but usually can not be visualized with light microscopy. Concern has been raised, however, that HRP reaction product, which is exquisitely sensitive when generated properly, may in some cases distribute to sites distant from the original binding of the conjugate and therefore result in spurious antigen localization.

Unlabeled Antibody Immunocytochemistry Applied to Murine Mammary Tumor Cells

1975 ◽  
Vol 33 ◽  
pp. 390-391
Author(s):  
Wm. J. Arnold ◽  
J. Russo ◽  
H. D. Soule ◽  
M. A. Rich
Keyword(s):  
Horseradish Peroxidase ◽  
Mammary Tumor ◽  
Covalent Binding ◽  
Petri Dish ◽  
Gamma Chain ◽  
Mammary Tumor Virus ◽  
Mammary Tumor Cells ◽  
Murine Mammary Tumor ◽  
Tumor Virus ◽  
Unlabeled Antibody

Our studies of mammary tumor virus have included the application of the unlabeled antibody enzyme method of Sternberger to mammary tumor derived mouse cells in culture and observation with an electron microscope. The method avoids the extravagance of covalent binding of indicator molecules (horseradish peroxidase) with precious antibody locator molecules by relying instead upon specific antibody-antigen linkages. Our reagents included: Primary Antibody, rabbit anti-murine mammary tumor virus (MuMTV) which was antiserum 113 AV-2; Secondary Antibody, goat anti-rabbit IgG gamma chain (Cappel Laboratories); andthe Indicator, rabbit anti-horseradish peroxidase - horseradish peroxidase complex (PAP) (Cappel Labs.). Dilutions and washes were made in 0.05 M Tris 0.15 M saline buffered to pH 7.4. Cell monolayers, after light fixation in glutaraldehyde, were incubated in place by a protocol adapted from Sternberger and Graham and Karnovsky, then embedded by our usual method for monolayers. Reagents were confined to specific areas by neoprene 0-rings (Parker Seal Co.) reducing the amount of reagent needed to 50 microliters, 1/6th of that required to wet a 35 mm petri dish.

Ultrastructural and immunocytochemical studies of the rat hypothalamo-neurohypophysial system processed by rapid freezing and freeze-substitution fixation

1990 ◽  
Vol 48 (3) ◽  
pp. 884-885
Author(s):  
Seiji Shioda ◽  
Yasumitsu Nakai ◽  
Atsushi Ichikawa ◽  
Hidehiko Ochiai ◽  
Nobuko Naito
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Wistar Rat ◽  
Neurosecretory Cells ◽  
Rapid Freezing ◽  
Sodium Metaperiodate ◽  
Tissue Sections ◽  
Ultrathin Sections ◽  
Electron Microscopes

The ultrastructure of neurosecretory cells and glia cells in the supraoptic nucleus (SON) of the hypothalamus and the neurohypophysis (PN) was studied after rapid freezing followed by substituion fixation. Also, the ultrastructural localization of vasopressin (VP) or its carrier protein neurophys in II (NPII) in the SON and PN was demonstrated by using a post-embedding immunoco1loidal gold staining method on the tissue sections processed by rapid freezing and freeze-substitution fixation.Adult male Wistar rat hypothalamus and pituitary gland were quenched by smashing against a copper block surface precooled with liquid helium and freeze-substituted in 3% osmium tetroxide-acetone solutions kept at -80°C for 36-48h. After substituion fixation, the tissue blocks were warmed up to room temperature, washed in acetone and then embedded in an Epon-Araldite mixture. Ultrathin sections mounted on 200 mesh nickel grids were immersed in saturated sodium metaperiodate and then incubated in each of the following solutions: 1 % egg albumin in phosphate buffer, VP or NPII (1/1000-1/5000) antiserum 24h at 4°C, 3) colloidal gold solution (1/20) 1h at 20°C. The sections were washed with distilled waterand dried, then stained with uranylacetate and lead citrate and examined with Hitachi HU-12A and H-800 electron microscopes.

scholarly journals The rosy locus in Drosophila melanogaster: xanthine dehydrogenase and eye pigments.

Genetics ◽  
1991 ◽  
Vol 129 (4) ◽  
pp. 1099-1109 ◽  
Author(s):  
A G Reaume ◽  
D A Knecht ◽  
A Chovnick
Keyword(s):  
Drosophila Melanogaster ◽  
Structural Integrity ◽  
Present Report ◽  
Specific Interaction ◽  
Pigment Granule ◽  
Ultrastructural Localization ◽  
Xanthine Dehydrogenase ◽  
Pigment Formation ◽  
Antibody Staining ◽  
Pigment Granules

Abstract The rosy gene in Drosophila melanogaster codes for the enzyme xanthine dehydrogenase (XDH). Mutants that have no enzyme activity are characterized by a brownish eye color phenotype reflecting a deficiency in the red eye pigment. Xanthine dehydrogenase is not synthesized in the eye, but rather is transported there. The present report describes the ultrastructural localization of XDH in the Drosophila eye. Three lines of evidence are presented demonstrating that XDH is sequestered within specific vacuoles, the type II pigment granules. Histochemical and antibody staining of frozen sections, as well as thin layer chromatography studies of several adult genotypes serve to examine some of the factors and genic interactions that may be involved in transport of XDH, and in eye pigment formation. While a specific function for XDH in the synthesis of the red, pteridine eye pigments remains unknown, these studies present evidence that: (1) the incorporation of XDH into the pigment granules requires specific interaction between a normal XDH molecule and one or more transport proteins; (2) the structural integrity of the pigment granule itself is dependent upon the presence of a normal balance of eye pigments, a notion advanced earlier.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF Mg++-DEPENDENT DINITROPHENOL-STIMULATED ADENOSINE TRIPHOSPHATASE IN HUMAN BLOOD PLATELETS

1967 ◽  
Vol 15 (5) ◽  
pp. 267-272 ◽  
Author(s):  
VICTOR G. VETHAMANY ◽  
SYDNEY S. LAZARUS
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Human Blood ◽  
Adenosine Triphosphatase ◽  
Blood Platelets ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Structural Localization ◽  
Adenosine Triphosphatase Activity ◽  
Human Blood Platelets

Fine structural localization of adenosine triphosphatase activity was studied in human platelets briefly fixed in cold formol calcium and then incubated in lead medium with added dinitrophenol. Under these conditions, the Mg++-dependent dinitrophenol-stimulated adenosine triphosphatase of platelet mitochondria was demonstrated, but neither granules nor plasma membrane showed enzyme activity.

scholarly journals ELECTRON MICROSCOPIC STUDY OF THE ADRENOCORTICOTROPIN-PRODUCING CELL WITH THE USE OF UNLABELED ANTIBODY AND THE SOLUBLE PEROXIDASE-ANTIPEROXIDASE COMPLEX

1972 ◽  
Vol 20 (8) ◽  
pp. 590-603 ◽  
Author(s):  
G. C. MORIARTY ◽  
N. S. HALMI
Keyword(s):  
Secretory Granules ◽  
Staining Intensity ◽  
Electron Microscopic ◽  
Early Effect ◽  
Acth Cell ◽  
Maximal Diameter ◽  
Soluble Peroxidase ◽  
Ultrathin Sections ◽  
Acth Secreting ◽  
Unlabeled Antibody

The technique involving use of unlabeled antibody and the peroxidase-antiperoxidase complex was used to identify the adrenocorticotropin (ACTH)-secreting cell in the anterior pituitary lobe of the rat and to localize ACTH in it electron microscopically in ultrathin sections. The ACTH cell is star-shaped, with processes extending around other cells, and contains secretory granules of a maximal diameter of 300 mµ arranged peripherally along the plasma membrane. Stain was observed on secretory granules, around them, in the Golgi complex and in rough endoplasmic reticulum. One day after adrenalectomy, the ACTH cell is degranulated and the staining intensity of its remaining granules and cytoplasm is decreased, suggesting release of ACTH stores. If cortisol is given 6 hr after adrenalectomy, 18 hr later the ACTH cells are well granulated and the granules stain more intensely than normal. In addition, staining around the granules and throughout the cytoplasm is more intense, suggesting that an early effect of cortisol is to block release of ACTH. Twenty-one days after adrenalectomy, the ACTH cells are greatly increased in numbers and have complex, tortuous processes filled with intensely stained secretory granules.

Ultrastructural localization of the small GTP-binding protein Rap 1 in human platelets and megakaryocytes

1994 ◽  
Vol 88 (2) ◽  
pp. 372-382 ◽  
Author(s):  
Gaëtan Berger ◽  
Rozenn Quarck ◽  
Danié;le Tenza ◽  
Sylviane Levy-Toledano ◽  
Jean de Gunzburg ◽  
...  
Keyword(s):  
Binding Protein ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Gtp Binding Protein ◽  
Gtp Binding
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