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PubMed1991

Timed acid bath effects on kidney podocytes prepared for scanning electron microscopy.

In perfusion fixation of dog kidney glomeruli, observation of tear fractured surfaces by scanning electron microscopy reveals the deposition of "debris" on the surface of glomerular visceral epithelium (podocytes) that is not deposited on other kidney tissue surfaces. It is of concern to remove this debris without damage to tissue surfaces. A 5 minute bath in 8N HCl (hydrochloric acid) following postfixation in 1% OsO4 (osmium tetroxide) removes only some of the debris. A 10 minute acid bath removes this debris without visible damage to podocyte surface structures such as cilia, plicae or blebs. A 20 minute acid bath is destructive to these surface features.

Animals↗
PubMed2005

Fixation of microsporidian spores for electron microscopy.

Fresh and frozen spores of the microsporidia Nosema apis and Nosema bombi were fixed using various fixatives at different times and temperatures. Paraformaldehyde and technical formaldehyde gave results comparable to or better than glutaraldehyde. Increased fixation temperature improved the fixation of spores from terrestrial hosts. Freezing did not destroy the cytology of the spore.

Microscopy, Electron↗
PubMed1997

Transcellular openings through frog microvascular endothelium.

Reconstructions from serial ultrathin sections of microvascular endothelium suggest that gaps, which are induced by a range of stimuli, may pass through endothelial cells as well as between them. To address the possibility that the transcellular gaps are not artefacts of aldehyde fixation, we have reconstructed fourteen gaps, induced by the ionophore A23187, in frog mesenteric microvessels where the primary fixative was osmium tetroxide. All fourteen gaps were transcellular. The different actions of osmium tetroxide and glutaraldehyde lead us to consider that it is highly unlikely that transcellular gaps are fixation artefacts.

Animals↗
PubMed1991

Microwave fixation provides rapid, primary fixation for light and electron microscopy and for immunohistochemistry and immunocytochemistry.

A relatively new approach to specimen preservation for morphologic studies uses microwave energy and chemicals. Microwave fixation can produce fixation results equal in quality to chemical fixation methods and equal in speed to freeze fixation methods. The importance of this microwave fixation technology lies in its potential to provide a standardized fixation approach in histopathology, immunohistochemistry, and immunocytochemistry.

Animals↗
PubMed1982

An effective fixative for glucocorticoid receptors in fetal tissues.

As a preliminary study in an autoradiographic study of glucocorticoid (GC) receptor localization in orofacial tissues of mouse fetuses, a search was made to determine the most effective fixative for preservation of the GC-receptor complex. Twelve-day-old mouse fetuses were administered tritiated triamcinolone acetonide (3H-TAC) intraamniotically and subsequently processed by one of the following three procedures: freeze-drying, prefixation with Karnovsky's fixative, or "the catechin fixative" (Karnovsky's fixative containing 1% D-catechin) and postfixation with osmium tetroxide. Light microscopic autoradiography and liquid scintillation counting of the specimens revealed that the catechin fixative gave the best results for fixation of the steroid-receptor complex and preservation of tissue structure. Light and electron microscopic autoradiographic studies of the time course of the localization of 3H-TAC in palatal shelves supported the catechin fixative as being the most effective in preservation of GC-receptor or ligand complexes.

Animals↗
PubMed1994

Blood-borne macromolecule induces FGF receptor gene expression after focal ischemia.

We have detected fibroblast growth factor receptor (FGFR) gene expression in the focal ischemia model. The FGFR gene expression in neurons can be explained by neuronal network disturbances, but the mechanism of astroglial gene expression remains uncertain. We speculated that blood-borne edema fluid may activate gene expression of astroglias. To prove this hypothesis, we compared the pattern's of gene expression of FGFR and distribution of edema fluid by using serial tissue sections of the middle cerebral artery (MCA) ischemia. The left MCA of twenty-four male Wistar rats were occluded, and sacrificed 1, 3, 4, 7 and 14 days later by transcardiac perfusion and fixation. The tissues were sliced thinly to 14 microns sections. Part of the tissue sections was used for in situ hybridization for rat FGFR with [35S]labeled RNA probes. The other part of the sections was used for immunostaining for albumin, immunoglobulin G (IgG) and IgM. The FGFR mRNA expression was evident in the lesion-side hemisphere. In the cortex, neurons mainly expressed FGFR gene in the cortex, whereas astroglias and capillary endothelium expressed FGFR in the corpus callosum and internal capsule. The albumin distributed cortex and white matter of the lesion-side and it extended to the contralateral side. The IgG distributed mainly in the lesion-side white matter, and in part extended to the contralateral side. The IgM only distribute to the infarcted area. When we compared topographical distribution of FGFR in the white matter and pattern of albumin, IgG and IgM distribution, pattern of IgG distribution correlated well to the area of FGFR expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

Hybrid external fixation in high-energy elbow fractures: a modular system with a promising future.

BACKGROUND: Severe, high-energy, periarticular elbow injuries producing a "floating joint" are a major surgical challenge. Their reconstruction and rehabilitation are not well documented. Therefore, the following reports our experience with treating such injuries caused by war wounds. METHODS: Seven adults with compound open peri- and intra-articular elbow fractures were treated in hybrid ring tubular fixation frames. After debridement, bone stabilization, and neurovascular reconstructions, early controlled daily movements were started in the affected joint. RESULTS: These seven patients had together seven humeral, five radial, and six ulnar fractures. All fractures united at a median time of 180 days. No deep infection developed. The functional end results assessed by the Khalfayan functional score were excellent in two, good in one, and fair in four of these severely mangled upper extremities. None was amputated. CONCLUSIONS: The Mangled Extremity Severity Score has been shown to be unable to provide a reliable assessment for severe high-energy limb injuries surgically managed with the modular hybrid thin wire tubular external fixation system. This hybrid system is a very useful addition to the surgical armamentarium of orthopedic trauma surgeons. It both allows complex surgical reconstructions and reduces the incidence of deep infections in these heavily contaminated injuries. The hybrid circular (thin wire) external fixation system is very modular and may provide secure skeletal stabilization even in cases of severely comminuted juxta-articular fractures on both sides of the elbow joint (floating elbow) with severe damage to soft tissues. This fixation system allows individual fixation of forearm bone fractures, thus allowing the preservation of pronation-supination movements.

Adult↗
PubMed1994

Fixation duress in the pathogenesis of upper eyelid retraction in thyroid orbitopathy. A prospective study.

BACKGROUND: Upper eyelid retraction in thyroid eye disease may be caused by proptosis, levator and Müller's muscle infiltration with fibrosis or individual fiber enlargement, excessive sympathetic innervation, abnormal adhesions between levator palpebrae muscle and surrounding tissues, or fixation duress. Fixation duress refers to upper eyelid retraction while fixating with an eye with inferior rectus muscle restriction due to excessive simultaneous firing of the ipsilateral superior rectus and levator palpebrae muscles. METHODS: The authors prospectively examined six patients with strabismus and thyroid eye disease associated with inferior rectus restriction and upper eyelid retraction in whom the eyelid retraction was suspected clinically to be a result of fixation duress. All six patients underwent recession of both inferior recti ranging from 3 to 6.5 mm (mean, 4.5 mm) to improve the ocular motility and alignment, reduce the diplopia, and mitigate the upper eyelid retraction. RESULTS: Reduction of upper eyelid retraction measured as the preoperative versus postoperative difference in corneal light reflex-upper eyelid margin measurements was achieved in all patients postoperatively, ranging from 1.5 to 5.5 mm (mean, 3.2 mm). Only one patient showed significant residual retraction to warrant consideration of upper eyelid surgery. CONCLUSION: Fixation duress plays a significant role in upper eyelid retraction of thyroid eye disease in a subset of patients with restriction of the inferior rectus muscle. In this selected group of patients, the upper eyelid retraction may be reduced or eliminated upon proper recession of the tight inferior rectus muscle(s).

Diplopia↗
PubMed1999

The use of chloromethyl-X-rosamine (Mitotracker red) to measure loss of mitochondrial membrane potential in apoptotic cells is incompatible with cell fixation.

BACKGROUND: A recent report by Macho et al. (Cytometry 25: 333-340, 1996) described the use of chloromethyl-X-rosamine (CMX-Ros) as a fixable probe for detection of loss of mitochondrial membrane potential (psi(mit)), an early event in many models of apoptosis. However, this previous report lacked a description of any direct comparisons between pre- and post-fixation analyses of normal and apoptotic cells stained with CMX-Ros. METHODS: Using a variety of cell types, we investigated the effect of paraformaldehyde fixation on cellular retention of CMX-Ros and the implications of this for the subsequent analysis of changes in psi(mit) in cells undergoing apoptosis. RESULTS: We found that following fixation, the resolution between normal cells with polarized mitochondria and apoptotic cells with depolarized mitochondria is reduced to the extent that accurate discrimination between the cell types is no longer possible. CONCLUSIONS: Overall, our results are consistent with CMX-Ros being a valid probe for psi(mit) in intact cells but only when the cells are stained and analyzed immediately. Thus, our results suggest that the proposed applications for CMX-Ros in multiple parameter analysis of fixed cells are inappropriate and will lead to spurious results.

Apoptosis↗
PubMed2003

Sperm ultrastructure in the nudibranch genus Halgerda with reference to other Discodorididae and to Chromodorididae (Mollusca: Opisthobranchia).

Comparative sperm ultrastructure within the molluscan nudibranch genus Halgerda (Discodorididae) was examined for the first time using transmission electron microscopy (TEM), based on 17 of the 35 known species. In addition, observations on two other discodorids are made to facilitate outgroup comparison with Halgerda, including one species of Discodoris (D. boholiensis) and Asteronotus cespitosus (currently accepted as the closest sister taxon to Halgerda). Comparison was also made with some genera of the Chromodorididae in view of sperm similarities. Spermatozoa of all species examined were of the complex, helical, elongate ( approximately 300-400 micro m) type characteristic of most heterobranch gastropods. These cells exhibit the following discrete regions (in anteroposterior sequence) : an acrosomal complex (composed of a rounded, membrane-bound vesicle and a column-like pedestal); a solid, helical nucleus; an elongate, helical midpiece (composed of an axoneme and associated nine coarse fibers, an enveloping mitochondrial derivative of matrix, and paracrystalline materials and glycogen helix); an annular complex; and a short glycogen piece. Of these regions, the midpiece is by far the longest, occupying over 90% of the total sperm length. Comparison with other members of the radula-bearing cryptobranch dorids reveals several sperm similarities to other genera in the clade, particularly those of other Discodorididae and also with the Chromodorididae. Comparison with previously studied genera reveals noteworthy sperm differences within the Discodorididae. The most notable differences are the internal structure of the acrosomal pedestal (long and homogeneous in Halgerda, Discodoris; short and homogeneous in Asteronotus; long and finely striated in Rostanga; oblong with angular electron-lucent striations in Jorunna) and the internal structure of the glycogen piece. The pronounced helical keels of most Halgerda and Discodoris nuclei contrast with the weakly helical nucleus of Asteronotus. Sperm features alone do not provide a means of defining the genus Halgerda or the family Discodorididae nor do they support the monophyletic status of the caryophyllidia-bearing dorids. Important sperm characters such as the acrosome, nucleus, and midpiece can often still be determined from specimens that have been initially fixed in formalin, then stored in ethanol for extended periods of time (i.e., museum material). Of all sperm features, the mitochondrial derivative of the midpiece is the most resistant to long-term fixation : the survival of acrosomal, nuclear, and axonemal components is variable, presumably a factor of prefixation autolysis, varied primary fixation times and temperatures, formalin quality, and duration of alcohol storage.

Animals↗
PubMed2005

Assessment of HOPE fixation in vitrectomy specimens in patients with chronic bilateral uveitis (masquerade syndrome).

BACKGROUND: Cytological examination of rapidly transported, unfixed vitreous specimens is considered the gold standard in exclusion of primary intraocular lymphoma (PIOL) in patients with idiopathic steroid resistant chronic uveitis. These specimens are difficult to interpret, and reports of "false negatives" or "false positives" are common. Fixation using HOPE solution (Herpes-glutamic acid buffer mediated Organic solvent Protection Effect) has been successfully applied in the investigation of cytospin preparations using immunocytology, in situ hybridisation and polymerase chain reaction (PCR). The purpose of this study was to compare the cytologic features of vitrectomy specimens from patients with clinical reactive vitritis and PIOL prepared following HOPE fixation with those in conventional cytospins and to identify any diagnostic pitfalls. METHODS: Pars plana vitrectomy was performed in 15 cases of patients with chronic uveitis. The vitreous samples were halved: one half was fixed in HOPE solution; the other half remained unfixed. All samples were subject to conventional staining, immunocytology and clonality analysis using polymerase chain reaction (IgH-PCR) and GeneScan. The specimens were assessed following by two pathologists who were masked to the cytological preparation method. The specimens were evaluated for cellularity, cellular appearance, cytoplasmic and nuclear features as well as quality of the immunostains. RESULTS: Twelve of the fifteen vitreous samples were diagnosed as reactive vitritis; in three specimens, a primary intraocular lymphoma of B-cell type was diagnosed. Compared to the unfixed vitreal specimens, the quality of the cytomorphology and immunocytology improved in the HOPE-fixed specimens. IgH-PCR and GeneScan analysis demonstrated polyclonal amplification products in the reactive cases, and monoclonal B-cell populations in the B-PIOL. CONCLUSION: Our results demonstrate that cytomorphology and immunoreactivity of vitreous specimens are well preserved following HOPE fixation. DNA of sufficient quality could be extracted from HOPE-fixed vitreous biopsies, in order to perform clonality analyses. HOPE fixation appears to be promising in simplifying the transportation of these specimens, and may improve the diagnostic reliability of vitreous specimens in patients with masquerade syndrome.

Adult↗
PubMed1995

S-100 beta has a neuronal localisation in the rat hindbrain revealed by an antigen retrieval method.

The localisation of S-100 in mammalian CNS neurons has been under debate for more than two decades. We address the question with two polyclonal and two new monoclonal antibodies. The specificity and the distribution in rat brain is based on an antigen retrieval method. We present evidence that aldehyde fixatives mask S-100 beta in neurons, and that the immunoreactivity is retrieved after trypsinisation. Neuronal S-100 beta is also detected in unfixed and ethanol fixed sections. The neuronal immunoreactivity is partly solubilised from unfixed tissue sections with 2.5 mM EDTA and is completely extracted with 2.5 mM EDTA and 1% Triton X-100. Most of the glial S-100 beta is washed out from unfixed tissue sections with saline. S-100 beta has distinct distribution in neurons of the hindbrain, i.e., the brainstem and cerebellum, but is not observed in the forebrain. One of the monoclonal antibodies immunostained neither neurons nor glia when it had been absorbed with S-100 crosslinked to nitrocellulose membranes. The distribution of neuronal S-100 beta differed from that of other neuronal calcium binding proteins, such as calbindin and parvalbumin. It was confined mainly to cholinergic neurons of the hindbrain. The presence of S-100 beta in distinct neuronal populations may indicate neurotrophic effects of S-100 beta. The notion is supported by the capability of S-100 to cause neurite outgrowth in vitro.

Animals↗
PubMed2006

EDAC fixation increases the demonstrability of biogenic amines in the unicellular Tetrahymena: a flow cytometric and confocal microscopic comparative analysis.

Earlier experiments demonstrated the presence of hormones of the higher ranked animals in Tetrahymena. In the present experiments two fixatives, paraformaldehyde, which is commonly used and a carbodiimide, EDAC that was recommended by Panula et al. for the immunocytochemistry of histamine, are compared in Tetrahymena for the demonstration of histamine and serotonin by using flow cytometry and confocal microscopy. Both hormone levels were significantly higher after EDAC fixation; serotonin almost doubled and histamine was more than fivefold. The confocal microscopic pictures were clearer and the hormones' localization was easier. The results support earlier observations on the presence of these hormones in Tetrahymena and points to the advantage of EDAC fixation for demonstrating these hormones immunocytochemically.

Animals↗
PubMed2005

Epoxy resin as fixative during freeze-substitution.

An alternative protocol for freeze-substitution is described. Araldite/Epon embedding medium (20% in acetone) is first used as a stabilizer (as e.g., OsO(4)) and then as embedding medium. The major components of the Araldite/Epon resin formulation react with proteins and lipids and provide for an excellent preservation and reasonable visualisation of the ultrastructure. The ultrastructural appearance can be deliberately influenced with the standard freeze-substitution procedure [Van Harreveld, A., Crowell, J., 1964. Electron microscopy after rapid freezing on a metal surface and substitution fixation. Anat. Rec. 149, 381-386.] using OsO(4) as stabilizing agent by protocols which degrade cytoplasmic and membrane proteins. Epoxy stabilized and embedded samples may become an important tool to get information about the effects of different reagents and protocols used in freeze-substitution. We believe that an in-depth understanding of the procedures is required to correctly interpret images and to complement studies of dynamic processes by light microscopy with reliable, highly detailed ultrastructural information. The block face of epoxy stabilized samples after ultrathin sectioning is highly suited for the analysis of the ultrastructure by AFM.

Animals↗
PubMed1998

Carnoy's fixative reduces the number of chymase-positive cells in immunocytochemical staining of cord-blood-derived human cultured mast cells.

KleinJan et al. (Allergy 1996;51:614-20) reported that Carnoy's fixative reduced the number of chymase-positive mast cells in the nasal mucosa. Therefore, in the present study, we investigated whether Carnoy's fixative reduces the number of chymase-positive cells from cord-blood-derived human cultured mast cells when compared with other types of fixatives. Human mast cells were obtained by culturing cord-blood-derived CD34-positive cells in the presence of stem cell factor and interleukin-6. Staining procedures of the cells in fixation with Carnoy's fixative and with other fixatives gave no differences among the number of tryptase-positive cells, whereas fixation with Carnoy's fixative for 15 min gave a significant decrease in the number of chymase-positive cells compared with acetone for 10 min. The number of chymase-positive cells decreased in a time-dependent manner under fixation with Carnoy's fixative, indicating that Carnoy's fixative had a negative effect on the number of chymase-positive cells from cord-blood-derived human cultured mast cells.

Acetic Acid↗
PubMed1994

High-frequency sonophoresis: permeation pathways and structural basis for enhanced permeability.

The mechanism of stratum corneum (SC) permeabilization by ultrasound (sonophoresis) is unknown. We studied here permeation pathways, and SC intercellular structural organization following applications of high-frequency sonophoresis to hairless mouse skin. Ruthenium tetroxide post-fixation and tracer solutions of LaNO3 and FITC-dextrans were employed to examine SC lamellar bilayers, lamellar body morphology and subcellular permeation pathways. Sonophoresis disrupted the compact organization of SC bilayers and LB-derived contents at the stratum granulosum (SG)-SC interface, leading to domain separation between 0 and 20 h, reverting by 48 h. Post-sonophoresis, tracers traversed the SC via lacunae within the lamellar bilayers, and via lamellae in sites that displayed domain separation. These studies provide insights about the penetration pathways, permeabilizing mechanisms, and kinetics of sonophoresis on the epidermis.

Animals↗
PubMed2003

An improved chemical fixation method suitable for immunogold localization of green fluorescent protein in the Golgi apparatus of tobacco Bright Yellow (BY-2) cells.

In plant systems, the green fluorescent protein (GFP) is increasingly used as a marker to study dynamics of the secretory apparatus using fluorescence microscopy. The purpose of this study was to immunogold localize the GFP, at the electron microscopic level, in a line of tobacco BY-2-cultured cells, expressing a GFP-tagged Golgi glycosyltransferase. To this end we have developed a simple, one-step chemical fixation method that allow good structural preservation and specific labeling with anti-GFP antibodies. Using this method, we have been able to show that an N-glycan GFP-tagged xylosyltransferase is specifically associated with Golgi stacks of BY-2 transformed cells and is preferentially located in medial cisternae. As an alternative to cryofixation methods, such as high-pressure freezing, which requires specialized and expensive equipment not available in most laboratories, this method offers researchers the opportunity to investigate GFP-tagged proteins of the endomembrane system in tobacco BY-2 cells.

Cell Wall↗