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.

Detection of p24 in HIV-1 infected cells embedded in LR White and Lowicryl K4M

1991 ◽  
Vol 23 (8) ◽  
pp. 381-384 ◽  
Author(s):  
Gregor Stransky ◽  
Robert F. Garry ◽  
Steffen Gay
Keyword(s):  
Lr White ◽  
Infected Cells ◽  
Lowicryl K4m ◽  
Hiv 1

scholarly journals The use of osmicated tissues for Lowicryl K4M embedding.

1992 ◽  
Vol 40 (6) ◽  
pp. 869-874 ◽  
Author(s):  
A Nanci ◽  
M Mazariegos ◽  
M Fortin
Keyword(s):  
Golgi Apparatus ◽  
Parotid Gland ◽  
Osmium Tetroxide ◽  
Immunogold Labeling ◽  
Potassium Ferrocyanide ◽  
Rat Incisor ◽  
Rat Parotid Gland ◽  
Ultrastructural Appearance ◽  
Rat Parotid ◽  
Lowicryl K4m

Tissue chopper slices of rat incisor, rat parotid gland, and chicken tibiae, fixed with 1% glutaraldehyde, were post-fixed with potassium ferrocyanide-reduced osmium tetroxide, dehydrated with methanol, and conventionally embedded in Lowicryl K4M at -20 degrees C. The tissues showed an ultrastructural appearance comparable with that of their Epon-embedded counterparts and, in particular, the Golgi apparatus was well defined. Furthermore, Lowicryl K4M-embedded osmicated tissues permitted both post-embedding lectin-gold cytochemistry and immunogold labeling.

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 Immunogold labeling of luciferase in the luminous bacterium Vibrio harveyi after fast-freeze fixation and different freeze-substitution and embedding procedures.

1989 ◽  
Vol 37 (5) ◽  
pp. 663-674 ◽  
Author(s):  
M T Nicolas ◽  
J M Bassot ◽  
G Nicolas
Keyword(s):  
Vibrio Harveyi ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Immunogold Labeling ◽  
Chemical Fixation ◽  
Luminous Bacterium ◽  
Immunogold Staining ◽  
Bioluminescent Bacterium ◽  
Lr White ◽  
Freeze Fixation

We studied the ultrastructural localization of luciferase on sections of the bioluminescent bacterium Vibrio harveyi by indirect immunogold staining, using a polyclonal antiluciferase antibody and the usual control tests, after chemical fixation or fast-freeze fixation (FFF) followed by different freeze-substitution (FS) procedures and embedding in either Epon or LR White. After liquid fixation with glutaraldehyde and paraformaldehyde and LR White embedding, labeling occurred over the cytoplasm but not over the condensed nucleoid. Epon embedding almost abolished it. FFF-FS considerably improved the morphological preservation and revealed cytoplasmic "patches" with a complex ultrastructure in Epon sections. The preservation was always less good in LR White. The patches were densely labeled, even in Epon sections, after FS in acetone. However, labeling intensity was 3.7 times greater in LR White than in Epon. With both resins, labeling diminished similarly when fixative agents were present in the FS medium. The localization of luciferase in the cytoplasm and particularly in the patches is discussed.

Further Observations on the Virus of Epizootic Diarrhea of Infant Mice. An Electron Microscopic Study

1967 ◽  
Vol 25 ◽  
pp. 110-111
Author(s):  
W. G. Banfield ◽  
G. Kasnic ◽  
J. H. Blackwell
Keyword(s):  
Electron Microscope ◽  
Infected Cell ◽  
Microscopic Study ◽  
Intestinal Epithelium ◽  
Ultrastructural Study ◽  
Osmium Tetroxide ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Virus Particles ◽  
Infected Cells

An ultrastructural study of the intestinal epithelium of mice infected with the agent of epizootic diarrhea of infant mice (EDIM virus) was first performed by Adams and Kraft. We have extended their observations and have found developmental forms of the virus and associated structures not reported by them.Three-day-old NLM strain mice were infected with EDIM virus and killed 48 to 168 hours later. Specimens of bowel were fixed in glutaraldehyde, post fixed in osmium tetroxide and embedded in epon. Sections were stained with uranyl magnesium acetate followed by lead citrate and examined in an updated RCA EMU-3F electron microscope.The cells containing virus particles (infected) are at the tips of the villi and occur throughout the intestine from duodenum through colon. All developmental forms of the virus are present from 48 to 168 hours after infection. Figure 1 is of cells without virus particles and figure 2 is of an infected cell. The nucleus and cytoplasm of the infected cells appear clearer than the cells without virus particles.

The Current Situation in Chemical Stabilization of Biological Materials

1972 ◽  
Vol 30 ◽  
pp. 132-133
Author(s):  
John H. Luft
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Molecular Level ◽  
Cross Linking ◽  
Chemical Stabilization ◽  
Specimen Preparation ◽  
Sufficient Condition ◽  
Radio Telescopes

With information processing devices such as radio telescopes, microscopes or hi-fi systems, the quality of the output often is limited by distortion or noise introduced at the input stage of the device. This analogy can be extended usefully to specimen preparation for the electron microscope; fixation, which initiates the processing sequence, is the single most important step and, unfortunately, is the least well understood. Although there is an abundance of fixation mixtures recommended in the light microscopy literature, osmium tetroxide and glutaraldehyde are favored for electron microscopy. These fixatives react vigorously with proteins at the molecular level. There is clear evidence for the cross-linking of proteins both by osmium tetroxide and glutaraldehyde and cross-linking may be a necessary if not sufficient condition to define fixatives as a class.

Immunogold localization of HMB-45 antigen in pulmonary lymphangiomyomatosis

1995 ◽  
Vol 53 ◽  
pp. 998-999
Author(s):  
J.M. Minda ◽  
E. Dessy ◽  
G. G. Pietra
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Muscle Cells ◽  
Ultrastructural Localization ◽  
Reproductive Age ◽  
Thin Sections ◽  
Smooth Muscle Proliferation ◽  
Hmb 45

Pulmonary lymphangiomyomatosis (PLAM) is a rare disease occurring exclusively in women of reproductive age. It involves the lungs, lymph nodes and lymphatic ducts. In the lungs, it is characterized by the proliferation of smooth muscle cells around lymphatics in the bronchovascular bundles, lobular septa and pleura The nature of smooth muscle proliferation in PLAM is still unclear. Recently, reactivity of the smooth muscle cells for HMB-45, a melanoma-related antigen has been reported by immunohistochemistry. The purpose of this study was the ultrastructural localization of HMB-45 immunoreactivity in these cells using gold-labeled antibodies.Lung tissue from three cases of PLAM, referred to our Institution for lung transplantation, was embedded in either Poly/Bed 812 post-fixed in 1% osmium tetroxide, or in LR White, without osmication. For the immunogold technique, thin sections were placed on Nickel grids and incubated with affinity purified, monoclonal anti-melanoma antibody HMB-45 (1:1) (Enzo Diag. Co) overnight at 4°C. After extensive washing with PBS, grids were treated with Goat-anti-mouse-IgG-Gold (5nm) (1:10) (Amersham Life Sci) for 1 hour, at room temperature.

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.

Child Diagnosis of Autism - How It Can Change the Family Life

2017 ◽  
Vol 5 (1) ◽  
pp. 498
Author(s):  
Maria Stănescu
Keyword(s):  
Early Intervention ◽  
Family Life ◽  
Social Life ◽  
Decisive Role ◽  
Autistic Children ◽  
Child Behaviors ◽  
High Quality ◽  
Child Diagnosis ◽  
The Family

The article is about the role of the family in the education and formation of children and, especially, in the life and development of autistic children. It describes the problems their family is facing and the need for counseling to parents with autistic children. The reaction to finding the diagnosis of autism varies from one family to another and may encounter a large variety: from disbelief, anger, guilt, helplessness, devastation, surprise, or even rejection of the child, to understanding and relief when finally the parents have an explanation for their child behaviors. Early intervention is important in psychological sustaining of the parent, as parent involvement in the recovery of the child with autism has a determinant role in his development and in ensuring a high quality of life of the child and the life of the hole family. The response to a child's autism diagnosis varies from one family to another. The family goes through a variety of disbelief, anger, guilt, helplessness, devastation, surprise, or even rejection of the child, to understanding and relief. Early intervention is very important in the psychological support of the parent. Because any change disturbs the family equilibrium. A diagnosis of autism changes not only the life of the diagnosed child, but also the life of family members. All the resources are focused on the need of the child. Although each parent is different, after diagnosing the child with autism, all parents are overwhelmed by confusion, shock and denial. Parents' feelings can be influenced by how their children's situation affects different aspects of life - it has an impact on service, on social life and all their personal life. If we look at the family as a system and when a disturbing factor appears, all parts of the system are affected. The involvement of parents in the recovery of the child with autism has a decisive role in its development and in ensuring a high quality of child's life and family life.

Perfusion fixation of lungs for structure-function analysis: credits and limitations

1982 ◽  
Vol 53 (2) ◽  
pp. 528-533 ◽  
Author(s):  
H. Bachofen ◽  
A. Ammann ◽  
D. Wangensteen ◽  
E. R. Weibel
Keyword(s):  
Osmium Tetroxide ◽  
Function Analysis ◽  
Transpulmonary Pressure ◽  
Uranyl Acetate ◽  
Vascular Perfusion ◽  
Lung Weight ◽  
Physiological Variables ◽  
Isotonic Buffer ◽  
Perfusion Fixation

The quality of tissue preservation in lungs fixed by vascular perfusion has been reevaluated. Excised rabbit lungs inflated to 60% of total lung capacity were perfused (zone III conditions) with different but widely used fixatives. The effects of the perfusates on pertinent physiological variables have been assessed by a continuous monitoring, the effects on the pulmonary microstructure by qualitative and morphometric analysis of electron micrographs. Important results include the following. 1) Perfusions with isotonic glutaraldehyde at flow rates within the physiological range produce large increases of perfusion pressure and lung weight that reflect intracellular, interstitial, and intra-alveolar edema. 2) No edema occurs if glutaraldehyde is added to isotonic buffer solutions (total osmolarity 510 mosM). 3) Glutaraldehyde as sole perfusate does not fully eliminate the retractive force of lung tissue. Upon release of transpulmonary pressure the lungs retract by an indeterminable amount. 4) Satisfactory results can be obtained by sequential perfusion with osmium tetroxide and uranyl acetate or glutaraldehyde (510 mosM) followed by osmium tetroxide and uranyl acetate. The latter combination yields optimal preparations to study the alveolar and capillary architecture but causes a hyperosmotic volume loss of lung cells (cell shrinkage).

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