scholarly journals Comparison of LR White and Unicryl as embedding media for light and electron immunomicroscopy of chromaffin cells.

1996 ◽  
Vol 44 (3) ◽  
pp. 289-295 ◽  
Author(s):  
G Goping ◽  
G A Kuijpers ◽  
R Vinet ◽  
H B Pollard
Keyword(s):  
Electron Microscopy ◽  
Tyrosine Hydroxylase ◽  
Membrane Fusion ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Osmium Tetroxide ◽  
Immunogold Labeling ◽  
Dopamine Beta Hydroxylase ◽  
Lr White ◽  
Embedding Medium

LR White and Unicryl are members of the same family of acrylic embedding resins and are very suitable for "on grid" postembedding immunogold labeling. We studied the ultrastructure of LR White- and Unicryl-embedded cultured chromaffin cells and the immunolocalization of three chromaffin cell proteins, the enzymes dopamine-beta-hydroxylase (DbetaH) and tyrosine hydroxylase (TH), and the membrane fusion and Ca2+ channel protein synexin (annexin VII). We report here that Unicryl is preferable to LR White as an embedding medium for electron microscopy when osmium tetroxide fixation is omitted. The basis for this distinction is better ultrastructural preservation and improved immunodetection efficiency.

scholarly journals Cellular localization of tyrosine hydroxylase by immunohistochemistry.

1975 ◽  
Vol 23 (1) ◽  
pp. 1-12 ◽  
Author(s):  
V M Pickel ◽  
T H Joh ◽  
P M Field ◽  
C G Becker ◽  
D J Reis
Keyword(s):  
Tyrosine Hydroxylase ◽  
Dopaminergic Neurons ◽  
Chromaffin Cells ◽  
Cellular Localization ◽  
Dopaminergic System ◽  
Noradrenergic Neurons ◽  
Dopamine Beta Hydroxylase ◽  
The Central Nervous System ◽  
Cervical Ganglia ◽  
Large Neurons

The enzyme tyrosine hydroxylase (TH) was immunohistochemically localized by the peroxidase-antiperoxidase method in rat to chromaffin cells of the adrenal medulla, large neurons and small darkly staining cells of the superior cervical ganglia and noradrenergic and dopaminergic neurons in brain. As compared with the conjugated peroxidase or immunofluorescence techniques, the peroxidase-antiperoxidase method gave the most selective and specific cytoplasmic localization of TH antisera in every tissue examined. The peroxidase staining with the TH antisera was more intense in dopaminergic than in noradrenergic neurons of the central nervous system. While TH was visualized in cell bodies of both dopaminergic and noradrenergic neurons, it could only be detected in axons and terminals in the dopaminergic system. The perikarya of noradrenergic neurons could be distinguished from dopaminergic neurons by the immunohistochemical demonstration of the enzyme dopamine-beta-hydroxylase only in the former.

NGF-like trophic support from peripheral nerve for grafted rhesus adrenal chromaffin cells

1990 ◽  
Vol 73 (3) ◽  
pp. 418-428 ◽  
Author(s):  
Jeffrey H. Kordower ◽  
Massimo S. Fiandaca ◽  
Mary F. D. Notter ◽  
John T. Hansen ◽  
Don M. Gash
Keyword(s):  
Tyrosine Hydroxylase ◽  
Adrenal Medulla ◽  
Sural Nerve ◽  
Chromogranin A ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Adrenal Chromaffin Cells ◽  
Content Type ◽  
Adrenal Chromaffin ◽  
Fine Print

✓ Autopsy results on patients and corresponding studies in nonhuman primates have revealed that autografts of adrenal medulla into the striatum, used as a treatment for Parkinson's disease, do not survive well. Because adrenal chromaffin cell viability may be limited by the low levels of available nerve growth factor (NGF) in the striatum, the present study was conducted to determine if transected peripheral nerve segments could provide sufficient levels of NGF to enhance chromaffin cell survival in vitro and in vivo. Aged female rhesus monkeys, rendered hemiparkinsonian by the drug MPTP (n-methyl-4-phenyl-1,2,3,6 tetrahydropyridine), received autografts into the striatum using a stereotactic approach, of either sural nerve or adrenal medulla, or cografts of adrenal medulla and sural nerve (three animals in each group). Cell cultures were established from tissue not used in the grafts. Adrenal chromaffin cells either cocultured with sural nerve segments or exposed to exogenous NGF differentiated into a neuronal phenotype. Chromaffin cell survival, when cografted with sural nerve into the striatum, was enhanced four- to eightfold from between 8000 and 18,000 surviving cells in grafts of adrenal tissue only up to 67,000 surviving chromaffin cells in cografts. In grafts of adrenal tissue only, the implant site consisted of an inflammatory focus. Surviving chromaffin cells, which could be identified by both chromogranin A and tyrosine hydroxylase staining, retained their endocrine phenotype. Cografted chromaffin cells exhibited multipolar neuritic processes and numerous chromaffin granules, and were also immunoreactive for tyrosine hydroxylase and chromogranin A. Blood vessels within the graft were fenestrated, indicating that the blood-brain barrier was not intact. Additionally, cografted chromaffin cells were observed in a postsynaptic relationship with axon terminals from an undetermined but presumably a host origin.

scholarly journals HYDROXYPROPYL METHACRYLATE, A NEW WATER-MISCIBLE EMBEDDING MEDIUM FOR ELECTRON MICROSCOPY

1965 ◽  
Vol 26 (1) ◽  
pp. 137-155 ◽  
Author(s):  
Elizabeth H. Leduc ◽  
S. J. Holt
Keyword(s):  
Electron Microscopy ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Rat Tissues ◽  
Enzymatic Extraction ◽  
Hydroxypropyl Methacrylate ◽  
Ultrathin Sections ◽  
Heat Polymerization ◽  
Embedding Medium ◽  
Fasted Rats

Aldehyde-fixed rat tissues were variously dehydrated and impregnated in water-miscible 2-hydroxypropyl methacrylate (HPMA) containing 3 to 20 per cent water and 0.1 per cent α,α-azobisisobutyronitrile as catalyst for subsequent polymerization with ultraviolet light. Heat polymerization was also effective. Blocks of embedded tissue readily gave ultrathin sections, which required staining by uranyl acetate and/or lead stains to give adequate contrast for electron microscopy. The ultrastructure of pancreas, kidney, muscle, and intestine was well preserved by aldehyde fixation alone. Use of postfixation in osmium tetroxide or direct osmium tetroxide fixation was unsatisfactory. The fine structure of aldehyde-fixed liver from fasted rats was well preserved, whereas that from normal rats showed considerable disorganization and collapse, apparently because of extraction of glycogen during the embedding procedure. Enzymatic extraction of proteins by pepsin and of ribonucleic acid by ribonuclease after either formaldehyde or glutaraldehyde fixation was rapidly effected by direct treatment of ultrathin sections with solutions of the enzymes. In contrast, no digestion of chromatin by deoxyribonuclease could be detected. In spite of this present limitation, HPMA appears to have several advantages over earlier water-miscible embedding media for electron microscopy and to be particularly suitable for ultrastructural cytochemistry.

scholarly journals Visualization of Identified GFP-expressing Cells by Light and Electron Microscopy

2003 ◽  
Vol 51 (3) ◽  
pp. 271-274 ◽  
Author(s):  
Katherine Luby-Phelps ◽  
Gang Ning ◽  
Joseph Fogerty ◽  
Joseph C. Besharse
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Immunogold Labeling ◽  
Living Tissue ◽  
Gfp Expression ◽  
Fixed Tissue ◽  
Lr White ◽  
Thin Sectioning ◽  
Gfp Fluorescence ◽  
Semithin Sections

We have developed a procedure for visualizing GFP expression in fixed tissue after embedding in LR White. We find that GFP fluorescence survives fixation in 4% paraformaldehyde/0.1% glutaraldehyde and can be visualized directly by fluorescence microscopy in unstained, 1-μm sections of LR White-embedded material. The antigenicity of the GFP is retained in these preparations, so that GFP localization can be visualized in the electron microscope after immunogold labeling with anti-GFP antibodies. The ultrastructural morphology of tissue fixed and embedded by this protocol is of quality sufficient for subcellular localization of GFP. Thus, expression of GFP constructs can be visualized in living tissue and the same cells relocated in semithin sections. Furthermore, semithin sections can be used to locate GFP-expressing cells for examination by immunoelectron microscopy of the same material after thin sectioning.

scholarly journals Ultrastructural localization of HTLV-I gag proteins p19 and p24 by single and double immunogold labeling.

1991 ◽  
Vol 39 (2) ◽  
pp. 185-191 ◽  
Author(s):  
G Stransky ◽  
S Gay
Keyword(s):  
Osmium Tetroxide ◽  
Decisive Role ◽  
Ultrastructural Localization ◽  
Immunogold Labeling ◽  
Gag Proteins ◽  
Lr White ◽  
Positive Immunostaining ◽  
Infected Cells ◽  
Lowicryl K4m

We developed a post-embedding immunogold labeling procedure for the ultrastructural localization of the HTLV-I gag proteins p19 and p24 by the use of monoclonal antibodies (MAb). Both antigens were shown to withstand fixation with 1% glutaraldehyde. In addition, p19 antigenicity was found not to be affected by post-fixation with 1% osmium tetroxide. The choice of resin played a decisive role in the retention of antigenicity. P19 was preserved in Lowicryl K4M as well as in LR White, whereas p24 was preserved only in Lowicryl. Both p19 and p24 were found to be localized on the HTLV-I virions themselves, whereas no positive immunostaining could be observed on the infected cells. In Lowicryl-embedded samples, in which both antigens had been preserved, a double immunogold labeling procedure was performed that allowed the co-localization of p19 and p24 on the same section. In osmicated LR White-embedded samples the quality of ultrastructural preservation of HTLV-I virions was found to be comparable to results obtained with the traditional glutaraldehyde-osmium tetroxide-epoxy resin processing.

Effects of substance P on the long-term regulation of tyrosine hydroxylase activity and catecholamine levels in cultured adrenal chromaffin cells

1986 ◽  
Vol 64 (12) ◽  
pp. 1548-1555 ◽  
Author(s):  
P. Boksa
Keyword(s):  
Tyrosine Hydroxylase ◽  
Substance P ◽  
Chromaffin Cells ◽  
Chromaffin Cell ◽  
Splanchnic Nerve ◽  
Adrenal Chromaffin Cells ◽  
Hydroxylase Activity ◽  
Tyrosine Hydroxylase Activity ◽  
Adrenal Chromaffin

Acetylcholine, released from splanchnic nerve terminals innervating adrenal chromaffin cells, is known to increase synthesis of adrenal tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. The neuropeptide substance P is also present in the splanchnic nerve innervating the adrenal medulla, and this study examined whether substance P has any long-term effects on tyrosine hydroxylase activity and catecholamine levels in cultures of adult bovine adrenal chromaffin cells. When cultures were incubated for 3 days with substance P and carbachol, a cholinergic agonist, substance P (10−6 M, and greater) completely inhibited the increase in tyrosine hydroxylase activity normally induced by carbachol. Long-term stimulation with carbachol also depleted endogenous catecholamines from the cells and substance P prevented this carbachol-induced depletion of catecholamine content. Substance P by itself, in the absence of carbachol, had only a slight effect on tyrosine hydroxylase activity. 8-Bromoadenosine 3′:5′-cyclic monophosphate, an analogue of adenosine 3′:5′-cyclic monophosphate, also increases tyrosine hydroxylase activity in chromaffin cells; however, substance P had no effect on the increase in tyrosine hydroxylase activity induced by this analogue. These results indicate that substance P's effects are relatively specific for the carbachol-induced increased in tyrosine hydroxylase activity and that the primary site of action of substance P is not a site common to the mechanism of tyrosine hydroxylase induction by carbachol and 8-bromoadenosine 3′:5′-cyclic monophosphate. During long-term incubation of chromaffin cell cultures, substance P inhibited the carbachol-induced increase in tyrosine hydroxylase activity only if peptidase inhibitors, bacitracin and captopril, were included in the medium, suggesting that chromaffin cell peptidases may be capable of terminating substance P's actions in these cells. The peptidase inhibitors bacitracin and captopril alone increased tyrosine hydroxylase activity and depleted catecholamines from the cells. The results suggest that substance P, released either from the splanchnic nerve or from the chromaffin cells themselves, may play a role in the long-term regulation of tyrosine hydroxylase activity and catecholamine levels in the mature adrenal chromaffin cell.

Spontaneous Cataract in Nude Athymic Mice

1977 ◽  
Vol 35 ◽  
pp. 512-513
Author(s):  
Ronald H. Bradley ◽  
R. S. Berk ◽  
L. D. Hazlett
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Nude Mouse ◽  
Cellular Immunity ◽  
Phosphate Buffer ◽  
Osmium Tetroxide ◽  
Athymic Mice ◽  
Transmission Microscopy ◽  
Mouse Lens ◽  
Scanning Electron

The nude mouse is a hairless mutant (homozygous for the mutation nude, nu/nu), which is born lacking a thymus and possesses a severe defect in cellular immunity. Spontaneous unilateral cataractous lesions were noted (during ocular examination using a stereomicroscope at 40X) in 14 of a series of 60 animals (20%). This transmission and scanning microscopic study characterizes the morphology of this cataract and contrasts these data with normal nude mouse lens.All animals were sacrificed by an ether overdose. Eyes were enucleated and immersed in a mixed fixative (1% osmium tetroxide and 6% glutaraldehyde in Sorenson's phosphate buffer pH 7.4 at 0-4°C) for 3 hours, dehydrated in graded ethanols and embedded in Epon-Araldite for transmission microscopy. Specimens for scanning electron microscopy were fixed similarly, dehydrated in graded ethanols, then to graded changes of Freon 113 and ethanol to 100% Freon 113 and critically point dried in a Bomar critical point dryer using Freon 13 as the transition fluid.

Scanning and Transmission Microscopy of Nematocyst Batteries in Epitheliomuscular Cells of Hydra

1971 ◽  
Vol 29 ◽  
pp. 410-411 ◽  
Author(s):  
Jane A. Westfall ◽  
S. Yamataka ◽  
Paul D. Enos
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Osmium Tetroxide ◽  
External Surface ◽  
Three Dimensional ◽  
Surface Structures ◽  
Transmission Microscopy ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Electron

Scanning electron microscopy (SEM) provides three dimensional details of external surface structures and supplements ultrastructural information provided by transmission electron microscopy (TEM). Animals composed of watery jellylike tissues such as hydras and other coelenterates have not been considered suitable for SEM studies because of the difficulty in preserving such organisms in a normal state. This study demonstrates 1) the successful use of SEM on such tissue, and 2) the unique arrangement of batteries of nematocysts within large epitheliomuscular cells on tentacles of Hydra littoralis.Whole specimens of Hydra were prepared for SEM (Figs. 1 and 2) by the fix, freeze-dry, coat technique of Small and Màrszalek. The specimens were fixed in osmium tetroxide and mercuric chloride, freeze-dried in vacuo on a prechilled 1 Kg brass block, and coated with gold-palladium. Tissues for TEM (Figs. 3 and 4) were fixed in glutaraldehyde followed by osmium tetroxide. Scanning micrographs were taken on a Cambridge Stereoscan Mark II A microscope at 10 KV and transmission micrographs were taken on an RCA EMU 3G microscope (Fig. 3) or on a Hitachi HU 11B microscope (Fig. 4).

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