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 Electron microscopic localization of chromogranin A in osmium-fixed neuroendocrine cells with a protein A-gold technique.

1987 ◽  
Vol 35 (7) ◽  
pp. 795-801 ◽  
Author(s):  
S A Hearn
Keyword(s):  
Neuroendocrine Tumors ◽  
Chromogranin A ◽  
Osmium Tetroxide ◽  
Secretory Granules ◽  
Protein A ◽  
Neuroendocrine Cells ◽  
Neurosecretory Granules ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Carotid Body Tumors

An antibody (LK2H10) to chromogranin A has been recommended for use in ultrastructural identification of neuroendocrine secretory granules. Previous studies have demonstrated immunoreactive chromogranin A in specimens prepared for electron microscopy by glutaraldehyde fixation only. In this study, the effect of specimen post-fixation by osmium tetroxide on post-embedding localization of chromogranin A was evaluated. Human tissues from benign endocrine glands, neuroendocrine tumors, and non-neuroendocrine tumors were post-fixed in osmium, embedded in epoxy resin, and the sample thin sections immunolabeled using a protein A-gold technique. Chromogranin A-positive neurosecretory granules were detected in pancreatic islets, adrenal medulla, stomach, ileum, anterior pituitary, and parathyroid. Mid-gut carcinoids, bronchial carcinoids, pheochromocytomas, paragangliomas, carotid body tumors, and thyroid medullary carcinomas contained immunoreactive granules. Cytoplasmic granules in non-neuroendocrine tumors did not react for chromogranin A. Tissues post-fixed in osmium tetroxide had optimally preserved ultrastructural features, and use of this fixative is compatible with postembedding localization of chromogranin A in neurosecretory granules.

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 Use of dinitrophenol-IgG conjugates to detect sparse antigens by immunogold labeling.

1989 ◽  
Vol 37 (1) ◽  
pp. 69-74 ◽  
Author(s):  
R K Pathak ◽  
R G Anderson
Keyword(s):  
Osmium Tetroxide ◽  
Low Density Lipoprotein ◽  
Protein A ◽  
Density Lipoprotein ◽  
Immunogold Labeling ◽  
Low Density ◽  
Good Sensitivity ◽  
Thin Sections ◽  
Permeabilized Cells ◽  
Novel Method

We describe a novel method for localizing sparse antigens in thin sections by protein A-gold labeling. The primary antibody is applied to fixed and detergent-permeabilized cells. The cells are then incubated with an anti-antibody that has been labeled with multiple dinitrophenol residues. The cells are next fixed again with glutaraldehyde and osmium tetroxide fixatives before embedding in Eponate. When thin sections are prepared, the dinitrophenol residues are readily detected with a tertiary anti-DNP antibody followed by protein A-gold labeling. This method offers good sensitivity along with superior morphology. Our test antigen for this method was the receptor for low-density lipoprotein, an antigen which had evaded detection by protein A-gold using ultra-thin cryosections.

The Effect of Electron Microscopy on Pathological Diagnosis of Neoplasm

1990 ◽  
Vol 48 (3) ◽  
pp. 248-249
Author(s):  
Xiaojun Zhou ◽  
Taihe Zhang
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Frozen Section ◽  
Protein A ◽  
Functional Classification ◽  
Lead Citrate ◽  
Thin Sections ◽  
Tumor Tissues ◽  
Frozen Section Diagnosis

Although electron microscopy (EM) has contributed enormously to an understanding of the structural intricacies of tumor cells, the usefulness of EM in pathological diagnoses of neoplasms has not been readily appreciated by general pathologists. In the present study, 223 tumors submitted for EM diagnosis were analyzed in an attempt to gain further information concerning the contribution of EM to tumor diagnosis.223 neoplasms were submitted to EM for their final diagnoses when histopathological diagnoses were obscure, which represented about of the total number of surgical tumor specimens. Most specimens were taken at the time of frozen section diagnosis and a small number of tissues were originally fixed informaldehyde. All of tissues were fixed with buffered glutaradehyde, postfixed with osmium tetroxide and embedded in Epon 812. Ultrasections were made after semith in sections were examined to verify that representative tumor tissues were present. Thin sections were stained with uranium acetate and lead citrate, and examined under JEM-1200 EX electron microscope. In selected cases, mainly with neuroendocrine tumors, nickel grid-mounted sections were subjected to post embedding immunoelectron microscopy (IEM) using protein A-gold for more detailed functional classification. Protein A-gold probes were prepared as Wang and co-workers described.

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.

Electron spectroscopic imaging for high-resolution immunocytochemistry: use of boronated protein A.

1989 ◽  
Vol 37 (5) ◽  
pp. 573-580 ◽  
Author(s):  
M Bendayan ◽  
R F Barth ◽  
D Gingras ◽  
I Londoño ◽  
P T Robinson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
High Resolution ◽  
Binding Sites ◽  
Secretory Granules ◽  
Protein A ◽  
Spectroscopic Imaging ◽  
Acinar Cells ◽  
Indirect Detection ◽  
Electron Spectroscopic Imaging ◽  
Thin Sections

In the present study we adapted electron spectroscopic imaging (ESI) for high-resolution immunocytochemistry. To accomplish this, we applied boronated protein A (B-pA) for indirect detection of specific antigenic sites using pre-embedding and post-embedding protocols. Isolated acinar cells were exposed to wheat germ agglutinin (WGA) and anti-WGA, followed by B-pA, to reveal WGA binding sites at the level of the plasma membrane. The cells were then embedded in Epon and unstained ultra-thin sections were examined by electron microscopy using the ESI mode. For post-embedding, ultra-thin sections of glutaraldehyde-fixed, Lowicryl-embedded pancreatic tissue were exposed to specific antibodies (anti-insulin or anti-amylase), followed by B-pA. The unstained sections were examined using the ESI mode. In both cases, boron was detected with high resolution either at the level of the plasma membrane of acinar cells, demonstrating WGA binding sites, or over secretory granules in pancreatic insulin-secreting cells or acinar cells, demonstrating insulin and amylase, respectively. These findings were compared to those obtained with the protein A-gold technique, and have demonstrated the analogy of both types of labeling. In addition, several control experiments assessed this novel approach. They have demonstrated the specificity of labeling and the high reactivity of B-pA, as well as its antibody-binding properties. Finally, electron energy loss spectral analysis confirmed the presence of boron in the tissue sections at sites where immunolabeling was detected. These results demonstrate that ESI is an appropriate approach for cytochemistry. Since the technique is based on detection of elements, spatial resolution is considered to be in the magnitude of 0.5 nm, which represents a major improvement in resolution over actual electron microscopic cytochemical techniques.

scholarly journals Ultrastructural immunolocalization of elastic fibers in rat blood vessels using the protein A-gold technique.

1987 ◽  
Vol 35 (1) ◽  
pp. 15-21 ◽  
Author(s):  
C Lethias ◽  
D J Hartmann ◽  
M Masmejean ◽  
M Ravazzola ◽  
I Sabbagh ◽  
...  
Keyword(s):  
Blood Vessels ◽  
Protein A ◽  
High Sensitivity ◽  
Ultrastructural Level ◽  
Rat Aorta ◽  
Elastic Fibers ◽  
Thin Sections ◽  
Rat Blood ◽  
Human Dermis ◽  
Arteries And Veins

Identification of elastic fibers at the ultrastructural level is accomplished by a post-embedding immunohistochemical technique using the protein A-colloidal gold method. Antisera against elastins from human dermis and rat aorta have been characterized by radioimmunoassay and then applied to thin sections of rat blood vessels. Two fixative solutions and two embedding media have been tested. Both antibodies bind to elastic fibers of normal arteries and veins, indicating crossreactions among organs and species. The high sensitivity of this method is demonstrated by its application to the detection of neo-elastogenesis in the intimal thickening of aortic grafts.

scholarly journals Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique.

1983 ◽  
Vol 31 (1) ◽  
pp. 101-109 ◽  
Author(s):  
M Bendayan ◽  
M Zollinger
Keyword(s):  
Secretory Granules ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Liver Mitochondria ◽  
Secretory Proteins ◽  
Additional Advantage ◽  
Sodium Metaperiodate ◽  
Thin Sections ◽  
Antigenic Sites ◽  
Structural Preservation

The protein A-gold immunocytochemical technique has been modified to allow labeling of cellular antigenic sites on osmium-fixed or postfixed tissues. Several strong oxidizing agents have been found able to restore protein antigenicity on osmicated tissue thin sections. According to the fine structural preservation and intensities of labeling, pretreatment with sodium metaperiodate gave optimal results. Pancreatic secretory proteins (and/or proproteins) as well as insulin (and/or proinsulin) were localized over perfectly preserved rough endoplasmic reticulum (rER), Golgi apparatus, and secretory granules of the corresponding pancreatic cells; carbamyl phosphate synthetase and catalase were revealed over liver mitochondria and peroxisomes, respectively. In addition to the higher resolution in the labeling obtained using osmium-fixed tissues, the present modification confers an additional advantage to the protein A-gold technique by allowing labeling on tissues processed for routine electron microscopy.

scholarly journals A novel methodology for double protein A-gold immunolabeling utilizing the monovalent fragment of protein A.

1992 ◽  
Vol 40 (3) ◽  
pp. 435-441 ◽  
Author(s):  
J R Thorpe
Keyword(s):  
Electron Microscopy ◽  
Binding Sites ◽  
Secretory Granules ◽  
Protein A ◽  
Model System ◽  
Normal Serum ◽  
Gold Complex ◽  
Thin Sections ◽  
Igg Binding ◽  
Gold Probe

A method for sequential protein A-gold immunolabeling is described whereby the binding of second gold probe to the first antibody-protein A-gold complex is reduced to acceptably minimal levels. Immunolabeling of thin sections of embedded pituitary tissue was used as a model system. After an initial immunolabeling for prolactin, sections were incubated in normal serum (rabbit) followed by a monovalent fragment of protein A. These latter two incubations reduced artifactual second gold probe label over prolactin-labeled secretory granules to minimal levels (much less than 1 particle per granule) when sections were subsequently immunolabeled with normal serum. The combination of normal serum and protein A fragment incubations saturates IgG and protein A binding sites on the first antibody-gold probe complex. The latter is thereafter unable to bind further IgG (and thus gold probe) because of the monovalent nature of the protein A fragment. It is suggested that this methodology may be extended to multiple immunolabeling procedures for electron microscopy. In addition, when used before single labeling this method may be an effective way to minimize nonspecific IgG binding in cases where the tissue or antibody under study may be a problem.

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