scholarly journals Preembedding colloidal gold immunostaining of hypothalamic neurons: light and electron microscopic localization of beta-endorphin-immunoreactive perikarya.

1985 ◽  
Vol 33 (6) ◽  
pp. 499-507 ◽  
Author(s):  
R Lamberts ◽  
P C Goldsmith
Keyword(s):  
Immunoglobulin G ◽  
Colloidal Gold ◽  
Gold Deposition ◽  
Pituitary Cells ◽  
Electron Microscopic ◽  
Hypothalamic Neurons ◽  
Double Labeling ◽  
Thin Sections ◽  
Beta Endorphin ◽  
Rabbit Immunoglobulin

A preembedding immunogold staining (IGS) procedure was developed to identify beta-endorphin/adrenocorticotropic hormone immunoreactive neurons at the light and electron microscopic levels. Colchicine-treated rats were perfused with Nakane's periodate-lysine-paraformaldehyde fixative. Vibratome sections were incubated in primary antisera followed by goat anti-rabbit immunoglobulin G coupled to 16 nm colloidal gold, and, in some cases, rabbit immunoglobulin G coupled to gold. The appearance to pink to light red perikarya, corresponding to colloidal gold deposition at antigenic sites, was monitored under the light microscope. Positive cell bodies in the arcuate region sometimes extended lateral to the nucleus. Only proximal portions of neuronal processes were stained. At the ultrastructural level, colloidal gold labeled the periphery of 90-110 nm dense neurosecretory granules in the perikaryal cytoplasm and a few proximal axons. Clusters of gold particles, appearing free in the neuroplasm, actually labeled secretory granules in adjacent thin sections. Granules associated with the Golgi apparatus were not stained. Colloidal gold labeling of mature beta-endorphin granules, but not progranules, in rat hypothalamic neurons was confirmed using the peroxidase-antiperoxidase technique. The results correlate well with data on the intracellular processing of pro-opiomelanocortin in pituitary cells and prepropressophysin in the paraventricular nucleus. These data demonstrate the first application of the preembedding colloidal gold staining method for the identification of intracellular antigens within the central nervous system. The IGS method provides a definitive marker for single or double labeling of nervous tissue at both the light and electron microscopic levels.

scholarly journals Immunolabeling of adenohypophysial cells with protein A-colloidal gold--antibody complex for electron microscopy: use of the freeze-substitution technique in tissue preparation.

1984 ◽  
Vol 32 (7) ◽  
pp. 705-711 ◽  
Author(s):  
S Hisano ◽  
T Adachi ◽  
S Daikoku
Keyword(s):  
Colloidal Gold ◽  
Secretory Granules ◽  
Protein A ◽  
Freeze Substitution ◽  
Pituitary Cells ◽  
Tissue Preparation ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Cytoplasmic Matrix ◽  
Gold Particles

The value of the freeze-substitution (FS) method for preparing tissues for electron microscopic immunohistochemistry was studied by comparing anterior pituitary cells prepared by this method and by a conventional method. Ultrathin sections of tissues embedded in Epon were subjected to immunostaining. The antigens adrenocorticotropin (ACTH) and prolactin (PRL) in a single ultrathin section were demonstrated by a simple double-labeling technique using a protein A-colloidal gold-antibody (pAG-Ab) complex. The preservation of cellular ultrastructure was superior in preparations obtained by FS. Gold-labeling was seen over secretory granules, and in ACTH cells also over the cytoplasmic matrix. The labeling was more intense in preparations obtained by FS, judging from the numbers of gold particles. In the double-labeling procedure, in which the pA-small colloidal gold-anti-PRL complex and pA-large colloidal gold-anti-ACTH complex were applied sequentially to sections, no cross-labeling with small and large gold particles was observed. It is concluded that if the antisera are sufficiently specific, the use of FS and the pAG-Ab complex is very effective in peptide immunohistochemistry. However, in the double-labeling procedure it is essential that the Fc-binding sites of pAG are saturated by the use of excess amounts of antibodies.

scholarly journals Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

1992 ◽  
Vol 40 (11) ◽  
pp. 1647-1657 ◽  
Author(s):  
R W Dirks ◽  
A G Van Dorp ◽  
J Van Minnen ◽  
J A Fransen ◽  
M Van der Ploeg ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Lymnaea Stagnalis ◽  
Pond Snail ◽  
28S Rrna ◽  
Electron Microscopic ◽  
Rna Sequences ◽  
Double Labeling ◽  
Thin Sections ◽  
Cell Hormone

The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea stagnalis was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28S rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable preservation of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphology, but on Lowicryl sections this added fixative resulted in a decrease of label intensity. A variety of probes, including plasmids, PCR products, and oligonucleotides, were used and all provided good results, although the use of oligonucleotides on Lowicryl sections resulted in decreased gold labeling. The gold particles were found mainly associated with rough endoplasmic reticulum (RER) but were also observed in lysosomal structures. Finally, the in situ hybridization method presented in this study proved to be compatible with the immunocytochemical detection of the caudodorsal cell hormone, as demonstrated by double labeling experiments.

scholarly journals Coloidal gold, ferritin and peroxidase as markers for electron microscopic double labeling lectin techniques.

1978 ◽  
Vol 26 (3) ◽  
pp. 163-169 ◽  
Author(s):  
J Roth ◽  
M Binder
Keyword(s):  
Particle Size ◽  
Cell Surface ◽  
Quantitative Evaluation ◽  
Binding Sites ◽  
Colloidal Gold ◽  
Lectin Binding ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Optimal Amount ◽  
Lectin Binding Sites

Three markers, colloidal gold, ferritin and peroxidase, were checked for usefulness in double labeling of lectin-binding sites. The amount of various lectins for the stabilization of good sols of a different particle size was evaluated. Several lectin-gold complexes were prepared for electron microscopic labeling purposes, and the optimal amount of various lectins needed for stabilization of gold solutions of a different particle size was determined. The following combinations were investigated for their usefulness in labeling two different lectin-binding sites: lectin-gold and lectin-gold (different particle size), lectin-gold and lectin-ferritin, as well as lectin-ferritin and lectin-peroxidase. Of these combinations the latter did not give satisfactory results for double labeling. In all single and double labeling techniques with the above mentioned markers the quantitative evaluation of the number of lectin-binding sites is not feasible, but these techniques will be of considerable value for the investigation of the dynamics of different lectin-binding sites on the cell surface.

Colloidal Gold Markers: Preparation and Cell Labeling for Transmission and Scanning Electron Microscopy

1985 ◽  
Vol 43 ◽  
pp. 530-533
Author(s):  
Robert S. Molday
Keyword(s):  
Cell Surface ◽  
Colloidal Gold ◽  
Critical Evaluation ◽  
Cell Labeling ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Gold Particles ◽  
Backscattered Electrons ◽  
Wide Range ◽  
Scanning Electron

Colloidal gold particles have become one of the most widely used markers to detect, localize and, in some cases, quantitate cell surface and intracellular antigens and receptors since their introduction as transmission electron microscopic (TFM) markers by Faulk and Taylor in 1971 and as scanning electron microscopic (SEM) markers by Horisberger et al. in 1975. This interest in colloidal gold markers for cell labeling is based on their versatile properties for detection under the electron microscope. Colloidal gold particles are highly electron-dense which enables them to be seen under the TEM in thin sections of heavily stained cells. They can be prepared in a wide range of highly uniform sizes for visualization at different magnifications and for multiple labeling studies. Under the SEM, gold particles emit a high quantity of secondary electrons, backscattered electrons and characteristic X-ray signals and as a result, with the appropriate detectors, they can be readily distinguished from cell surface structures having a similar morphological appearance. The successful application of colloidal gold particles as markers for TEM and SEM however requires (i) careful preparation and characterization of both the gold markers and the ligand (protein)-gold conjugates, (ii) utilization of specific labeling techniques employing necessary controls to confirm the specificity of labeling, and (iii) critical evaluation of results in relation to the conditions used in labeling. These aspects of gold labeling will be considered here. Additional information can be obtained from recent reviews dealing specifically with gold markers and more generally with cell labeling techniques.

scholarly journals Electron microscopic immunocytochemistry. Silver enhancement of colloidal gold marker allows double labeling with the same primary antibody.

1986 ◽  
Vol 34 (10) ◽  
pp. 1337-1342 ◽  
Author(s):  
K Bienz ◽  
D Egger ◽  
L Pasamontes
Keyword(s):  
Colloidal Gold ◽  
Physical Development ◽  
Primary Antibody ◽  
Silver Enhancement ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Double Labeling ◽  
Gold Marker

Electron microscopic sections, immunocytochemically labeled with colloidal gold, can be prepared for double labeling by applying the "EM-silver enhancement" procedure. This method, a photographic, so-called physical, development, increases the size of the gold marker to a predeterminable value and thereby inactivates the anti-species antibody present on the gold grain, thus allowing the labeling of a second antigen with antibody raised in the same species.

scholarly journals Catecholamine innervation of enkephalinergic neurons in guinea pig hypothalamus: demonstration by an in vitro autoradiographic technique combined with a post-embedding immunogold method.

1988 ◽  
Vol 36 (5) ◽  
pp. 533-542 ◽  
Author(s):  
V Mitchell ◽  
J C Beauvillain ◽  
P Poulain ◽  
M Mazzuca
Keyword(s):  
Guinea Pig ◽  
Ultrastructural Level ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Morphological Evidence ◽  
Osmic Acid ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Thin Sections

To study the relationship between the catecholamine (CA) nerve endings and the enkephalinergic cell bodies in the magnocellular dorsal nucleus (MDN) of guinea pig hypothalamus, double-labeling experiments were performed on the same tissue section at the electron microscopic level. An in vitro autoradiographic (ARG) method for [3H]-norepinephrine (NE) or [3H]-dopamine (DA) was combined with a post-embedding immunogold cytochemical technique for Met-enkephalin (Met-enk) in colchicine-treated animals. Hypothalamic slices (450 micrograms) were perfused with [3H]-NE or [3H]-DA at the fluid-gas interface, then fixed by immersion with glutaraldehyde and osmic acid. Semi-thin sections processed from the thickness of the slices showed adequate penetration of the tracers to all parts of the tissue. Frontal sections permitted visualization of some CA-uptake structures distributed around the cells. At the ultrastructural level, preservation appeared good on about 60% of the thickness of slices, and [3H]-CA structures were easily distinguished. Ultra-thin sections were successively incubated with Met-enk and colloidal gold-labeled antisera, followed by ARG processing. At the electron microscopic level, the good integrity of the tissue made possible visualization of [3H]-CA nerve terminals making synaptic contacts with enkephalinergic perikarya. These results provide morphological evidence for direct catecholaminergic control of enkephalinergic neurons of the MDN.

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.

The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

1973 ◽  
Vol 31 ◽  
pp. 574-575
Author(s):  
J. T. Stasny ◽  
R. C. Burns ◽  
R. W. F. Hardy
Keyword(s):  
Cellular Localization ◽  
Direct Evidence ◽  
Azotobacter Vinelandii ◽  
Intracellular Localization ◽  
Protein Component ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Fe Protein ◽  
Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

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