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.

scholarly journals Immunocytochemical evidence for vasopressin receptors.

1978 ◽  
Vol 26 (7) ◽  
pp. 581-592 ◽  
Author(s):  
M Castel
Keyword(s):  
Solid Phase ◽  
Secretory Granules ◽  
Cell Types ◽  
Staining Intensity ◽  
Pars Intermedia ◽  
Electron Microscopic ◽  
Neural Lobe ◽  
Acth Cell ◽  
Receptor Sites ◽  
Vasopressin Receptors

An electron microscopic study was made of mouse pituitaries immunocytochemically stained with anti-lysine vasopressin (LVP) as the primary antiserum in the unlabeled antibody peroxidase-anti-peroxidase procedure. Vasopressin (VP) was identified in the neurosecretory granules of the neural lobe which stained with peroxidase anti-peroxidase molecules. Electron density was induced in secretory granules of the pars intermedia (PI), both in the melanocyte stimulated hormone and ACTH cell types, probably indicating VP molecules attached to binding (receptor) sites. Omission of anti-LVP abolished staining both in the neural lobe and the PL Anti-LVP absorbed with antigen, by admixing with LVP, abolished staining in the neural lobe but not in the PI; according to optical density measurements the PI showed a +/- 22% staining increase over controls. Staining intensity in the PI probably reflects occupancy of binding (receptor) sites for VP. Exposure of PI granules to LVP before the usual staining sequence resulted in +/- 48% increased staining. In water-deprived mice with high endogenous VP titers, staining was +/- 33% and +/- 40% more intense than in normal mice. Solid phase absorbed and eluted antibodies to LVP provided additional proof that staining in both neural lobe and PI could be attributed to anti-LVP. Results indicate that binding or receptor sites for VP are located on secretory granules in the PL Possible physiological significance is discussed.

scholarly journals THE UNLABELED ANTIBODY ENZYME METHOD OF IMMUNOCYTOCHEMISTRY QUANTITATIVE COMPARISON OF SENSITIVITIES WITH AND WITHOUT PEROXIDASE-ANTIPEROXIDASE COMPLEX

1974 ◽  
Vol 22 (8) ◽  
pp. 782-801 ◽  
Author(s):  
JOHN P. PETRALI ◽  
DENNIS M. HINTON ◽  
GWEN C. MORIARTY ◽  
LUDWIG A. STERNBERGER
Keyword(s):  
Secretory Granules ◽  
Enzyme Reaction ◽  
Fold Increase ◽  
Immunocytochemical Staining ◽  
Intermediate Lobe ◽  
Reaction Products ◽  
Electron Microscopic ◽  
Specific Staining ◽  
Enzyme Method ◽  
Unlabeled Antibody

Araldite sections of rat pituitary intermediate lobe were used with anti-17-39 adrenocorticotropin in the unlabeled antibody enzyme method to compare electron microscopic immunocytochemical staining by peroxidase-antiperoxidase complex (PAP) with antiserum or purified antibody to peroxidase followed by peroxidase (PO). Quantitation of normalized optical densities of secretory granules offered high significance comparison (P < 0.0001) of experimental with control values and of experimental values with each other. Use of purified antibody (prepared by a new density gradient method) yielded four times higher sensitivity than antiserum to PO, while a 6.5-fold increase would have been expected from the degree of contamination of anti-PO in the serum by nonanti-PO immunoglobulin. Use of PAP was four to five times more sensitive than purified anti-PO and 20 times more sensitive than antiserum to PO. The increased sensitivity of PAP is explained by the high over-all binding affinity of PO for anti-PO in the cyclic PAP molecule, thus preventing the losses of PO that occur during washing when anti-PO and PO have been applied in sequence. Identification of the characteristic, cyclic PAP molecules affords confirmation of specific staining at high resolution. In the sequential application of anti-PO and PO, no PAP molecules are formed, thus making distinction of specific from nonspecific deposition of enzyme reaction products ambiguous. With the use of anti-17-39 ACTH and the intermediate lobe, the unlabeled antibody enzyme method was 16,000-100,000 times more sensitive than radioimmunoassay.

scholarly journals ULTRASTRUCTURAL IMMUNOCYTOCHEMISTRY WITH UNLABELED ANTIBODIES AND THE PEROXIDASE-ANTIPEROXIDASE COMPLEX A TECHNIQUE MORE SENSITIVE THAN RADIOIMMUNOASSAY

1973 ◽  
Vol 21 (9) ◽  
pp. 825-833 ◽  
Author(s):  
GWEN C. MORIARTY ◽  
C. MICHAEL MORIARTY ◽  
LUDWIG A. STERNBERGER
Keyword(s):  
Staining Intensity ◽  
Poor Quality ◽  
Immunocytochemical Staining ◽  
Great Promise ◽  
Electron Microscopic ◽  
Immunocytochemical Technique ◽  
Titration Curves ◽  
Rat Pituitary ◽  
Normal Rat ◽  
Unlabeled Antibody

Titration curves were developed with antisera to 17-39ACTH (adrenocorticotropin) and 1-39ACTH with the techniques of radioimmunoassay and electron microscopic immunocytochemistry. For the latter method, the unlabeled antibody peroxidase-antiperoxidase complex immunocytochemical technique was used to stain normal rat pituitary intermediate lobes. By radioimmunoassay standards, the 17-39ACTH antiserum was of poor quality. Its titration curve exhibited a flat slope and it did not bind a significant amount of labeled antigen beyond a 1: 30 dilution. However, immunocytochemical staining was detected with this antiserum at dilutions as high as 1:1,500. The antiserum to 1-39ACTH was of better quality by radioimmunoassay standards. It bound 45% of the labeled antigen at a dilution of 1:5,000. Immunocytochemical staining intensity was nearly maximal at a 1:5,000 dilution and decreased progressively to a limiting value at 1:16,000. However, when incubation times in the antisera were increased from 3 min to match those of the radioimmunoassay (48 hr) maximal staining was achieved at dilutions as great as 1:512,000 where only trace amounts of the labeled antigen were bound in the radioimmunoassay. It was concluded that the unlabeled antibody peroxidase-antiperoxidase complex immunocytochemical technique was sensitive enough to detect antibodies in sera of low titer and/or avidity which are not detected by a radioimmunoassay. The technique holds great promise for a sensitive assay system.

IMMUNOHISTOCHEMICAL LOCALISATION OF SEROTONIN IN RABBIT PLATELETS

1987 ◽  
Author(s):  
M Kanzaki ◽  
H Kimura ◽  
J Ochi
Keyword(s):  
Dense Body ◽  
Picric Acid ◽  
Electron Microscopic Observation ◽  
Staining Intensity ◽  
Mouse Serum ◽  
Storage Site ◽  
Electron Microscopic ◽  
Biochemical Measurements ◽  
Ultrathin Sections ◽  
Triton X

Although it has been accepted that the dense bodymay be the most predominant storage site of serotonin (5HT), some literatures suggested that 5KT was localized more abundantly in the alpha-granules or theplasma membrane than in the dense body. The discrepancy may be due to different methods used.For example, the former dense body theory is mostly based on biochemical measurements of 5HT which may easily diffusible among subcellular fractions, and the latter hypothesis is proposed by autoradiographic demonstration of exogenously applied 5HT. In the present study, endogenous 5HT has been visualized by immunoelectron microscopy using monoclonal antibody against 5HT.Platelet rich plasma (PRP) of rabbits was suspended for 30 min in a fixative containing 0.5% glutaraldehyde, 4% paraformaldehyde and 0.5% picric acid in 0.1M phosphate buffer (PB; pH 7.4), and resuspended overnight in the glutaraldehyde-free fixative. After wash the PRP was incubated for 3 days with PB containing saline and 0.03% Triton X-100 (PBST), and reacted for 3 days with monoclonal 5HT antibody ( 1μg/ml). The immunoreactive sites were rendered visible byABC immunohistochemistry (ABC from Vector Co. USA) with DAB precipitation. The colorized PRP was osmificated (1%) for 30 min, dehydrated with alcohol, embedded in Spurr and cut into ultrathin sections for electron microscopic observation. For immunohistochemical controls monoclonal 5HT antibody preabsorbed with O.lmM 5HT or non-immune normal mouse serum was used as the primary antibody, and no specific reaction was observed. Very fine 5HT-positive immunoreaction products were clearly localized in some granules with different staining intensity. These positive granules were mostly round or ovoid in shape with variousdiameters. The present immunohistochemical results appears to support previous results suggesting that 5HT is located in such granules as alpha-granules anddense bodies.

Cis-dichloro-diammine platinum (II): a stain for electron microscopic preparations

1978 ◽  
Vol 36 (2) ◽  
pp. 122-123
Author(s):  
Ernst Heinen
Keyword(s):  
Heart Muscle ◽  
Secretory Granules ◽  
Cell Types ◽  
Electron Microscopic ◽  
Antimitotic Agent ◽  
Dense Granules ◽  
Pancreatic Cells ◽  
Ultrathin Sections ◽  
Very High

We have previously reported that cis-dichloro-diammine platinum (II) (cis-Pt), an antimitotic agent discovered by Rosenberg and coworkers, can be used to contrast ultrathin sections. It was interesting to tend to increase the contrast given by this cis-Pt staining and to establish to which radicals cis-Pt binds in the cell.We have analysed various cell types (fibroblasts and Ehrlich tumour cells cultivated in vitro or various tissues : liver, pancreas, heart muscle, epididyme, intestine, etc.) fixed in glutaraldehyde, embedded in Epon and stained by immersion in a cis-Pt solution (1 mg/ml, 37°C, 2 or 3 days, in darkness).In all cases chromatin, especially heterochromatin, presents a very high contrast after cis-Pt staining and can easily be distinguished from the nucleolus (fig.). In the cytoplasm only ribosomes are well contrasted. The other organites are generally poorly stained, in some mitochondria, dense granules or filaments are apparent. Secretory granules containing proteins or mucopolysaccharides (basigranular or goblet cells of the intestine, mastocytes, pancreatic cells, etc.) are faintly stained by cis-Pt. In the heart muscle actin and myosin are only poorly contrasted. Cis-Pt suits thus for contrasting nucleic acids especially for differentiating DNA from RNA containing structures.

scholarly journals Four unlabeled antibody bridge techniques: a comparison.

1981 ◽  
Vol 29 (12) ◽  
pp. 1397-1404 ◽  
Author(s):  
P Ordronneau ◽  
P B Lindström ◽  
P Petrusz
Keyword(s):  
Horseradish Peroxidase ◽  
Gamma Globulin ◽  
Staining Intensity ◽  
Reaction Products ◽  
Electron Microscopic ◽  
Microscopic Studies ◽  
Immunocytochemical Techniques ◽  
Unlabeled Antibody ◽  
Shape And Size

Four unlabeled antibody immunocytochemical techniques, the "single bridge" (Avrameas S: Immunocytochemistry 6:825, 1969; Mason TE, Phifer RF, Spicer SS, Swallow RS, Dreskin RD: J Histochem Cytochem 17:190, 1969a; Sternberger LA, Cuculis JJ: 1969), the "single peroxidase-antiperoxidase (PAP)" (Sternberger LA, Hardy PH Jr, Cuculis JJ, Meyer HG: J Histochem Cytochem 18:315, 1970), the "double PAP" (Vacca LL, Rosario SL, Zimmerman EA, Tomashefsky P, Ng P-Y, Hsu KC: J Histochem Cytochem 23:208, 1975) and the "double bridge" (Ordronneau P, Petrusz P: Am J Anat 158:491, 1980) were compared at both the light and electron microscopic levels. The "double" procedures involved repeating incubations with the bridge antibody, in this case, sheep anti-rabbit gamma globulin, followed either by a second PAP step for the "double PAP" or a second anti-horseradish peroxidase step and a single incubation in horseradish peroxidase for the "double bridge." At both the light and electron microscopic levels the staining intensity was greater with the "double" techniques than with the "single" ones. This is probably due to amplification achieved with the second sheep anti-rabbit gamma globulin step, permitting an increase in the number of horseradish peroxidase molecules bound for each molecule of tissue-bound primary antibody. Also, the quality of the various commercial PAP preparations tested was variable. With the weaker ones the staining intensity could be increased by performing an incubation in fresh horseradish peroxidase after the PAP step. Finally, in electron microscopic studies, the reaction products formed in both the bridge and PAP procedures were identical in shape and size.

scholarly journals Immunocytochemical localization of a folicle stimulating hormone-like molecule in the testis.

1977 ◽  
Vol 25 (10) ◽  
pp. 1119-1126 ◽  
Author(s):  
J C Hutson ◽  
P J Gardner ◽  
G C Moriarty
Keyword(s):  
Germ Cells ◽  
Follicle Stimulating Hormone ◽  
Staining Intensity ◽  
The Body ◽  
Nuclear Staining ◽  
Adult Rat ◽  
Ultrathin Sections ◽  
Immunocytochemical Studies ◽  
Unlabeled Antibody ◽  
Stimulating Hormone

A follicle-stimulating hormone (FSH)-like molecule was localized in normal adult rat testes as well as testosterone-treated hypophysectomized rat tests with an unlabeled antibody (anti-FSH), peroxidase-antiperoxidase complex technique. Anti-FSH bound specifically to ultrathin sections of acrosomes of spermatids and intranuclear bodies of early spermatids. Quantitation of staining intensity demonstrated that FSH, used as an absorbing antigen, would significantly reduce this binding. There was less anti-FSH binding to the acrosomes of spermatozoa in the body and tail of the epididymis as compared to the less mature germ cells located in the testis and head of the epididymis. The acrosomal and nuclear staining of spermatids taken from hypophysectomized animals was similar to staining observed in sham injected animals. Taken together, these results suggest that there is a molecule within the acrosome that is immunologically similar to FSH. Most importantly, these results emphasize the importance of conducting physiologic experiments in conjunction with immunocytochemical studies.

scholarly journals Immunocytochemical localization of renin in the submandibular gland of the mouse.

1978 ◽  
Vol 26 (10) ◽  
pp. 855-861 ◽  
Author(s):  
E Gresik ◽  
A Michelakis ◽  
T Barka ◽  
T Ross
Keyword(s):  
Submandibular Gland ◽  
Adult Mouse ◽  
Secretory Granules ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Light Microscopic Level ◽  
Electron Microscopic ◽  
Granular Convoluted Tubule ◽  
Enzyme Method ◽  
Unlabeled Antibody

Renin was localized in the submandibular gland of the adult mouse at light and electron microscopic levels by the unlabeled antibody enzyme method of Sternberger. At the light microscopic level, renin was confined to the granular convoluted tubule (GCT) segment of the gland with considerable variation among GCT cells in intensity of staining. Some GCT cells failed to stain for renin. The pattern of staining was the same in the gland of male and female mice, but in the glands of females GCT segments were smaller and less numerous. At the electron microscopic level, staining for renin was also confined to the GCT cells, and was localized exclusively to the secretory granules. The intensity of staining of the secretory granules within a given GCT cell varied; some cells contained only minimally reactive or negative secretory granules. All other organelles within the GCT cell, except condensing vacuoles, failed to stain.

Ultrastructural characterization of human corticotrophs (ACTH cells)

1982 ◽  
Vol 101 (4) ◽  
pp. 484-490 ◽  
Author(s):  
Masaru Tsumuraya ◽  
Toru Kameya
Keyword(s):  
Cell Shape ◽  
Secretory Granules ◽  
Ultrastructural Observation ◽  
Electron Microscopic ◽  
Acth Cell ◽  
Ultrastructural Characterization ◽  
Rat Pituitary ◽  
The Relationship ◽  
Granule Properties

Abstract. Ultrastructural characterization of human corticotrophs (ACTH cells) was performed by 'superimposition technique', which enabled detailed ultrastructural observation of immunoreactive ACTH cells in adjacent semi-thin light microscopic immunoperoxidase and routine electron microscopic sections. The human corticotrophs were large and round or polygonal and were not stellate. They had scanty rough endoplasmic membranes and were packed with numerous large secretory granules measuring from 250 to 500 nm in diameter. The sizes of secretory granules in 6 human pituitaries were 448 ± 128, 344 ± 86, 448 ± 117, 244 ± 65, 316 ± 76, and 340 ± 93 nm, respectively. The granules were not seen in a single row along the plasma membrane as is the case in the rat. They possessed somewhat irregular outlines with a rarely discernible halo. Different densities of granule matrices were occasionally found. The cells often contained a few large heterogeneous vacuoles. From these findings, the human ACTH cells were recognized to be remarkably different in cell shape and size, properties of secretory granules and cytoplamic inclusions from those of the rat pituitary gland. In respect to secretory granule properties, the human ACTH cells are similar to those of some other mammals (fox, young pig, and lerot). More data is required to elucidate the relationship between human ACTH cell morphology and functional state.

Ultrastructural Aspects of Golgi Complex Function in Parathyroid Cells of Winter Frogs

1973 ◽  
Vol 31 ◽  
pp. 680-681
Author(s):  
William J. Dougherty
Keyword(s):  
Golgi Complex ◽  
Secretory Granules ◽  
Complex Function ◽  
Secretory Activity ◽  
Secretory Proteins ◽  
Perivascular Spaces ◽  
Pituitary Cells ◽  
Electron Microscopic ◽  
Ca Ions ◽  
Regulation Of Secretion

The regulation of secretion in exocrine and endocrine cells has long been of interest. Electron microscopic and other studies have demonstrated that secretory proteins synthesized on ribosomes are transported by the rough ER to the Golgi complex where they are concentrated into secretory granules. During active secretion, secretory granules fuse with the cell membrane, liberating and discharging their contents into the perivascular spaces. When secretory activity is suppressed in anterior pituitary cells, undischarged secretory granules may be degraded by lysosomes. In the parathyroid gland, evidence indicates that the level of blood Ca ions regulates both the production and release of parathormone. Thus, when serum Ca is low, synthesis and release of parathormone are both stimulated; when serum Ca is elevated, these processes are inhibited.

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