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At least 1,117 records · Page 62Linked to original sources
PubMed1995

Efferent neurons to the macular lagena in the embryonic chick.

The lipophilic dye, DiI, was placed into the macula lagena of paraformaldehyde-fixed embryonic chicks. Retrogradely labeled cells were found bilaterally in the pontine reticular formation (RF) between the dorsal facial nucleus and the abducens nerve root. This location is similar to that of the dorsomedial group of efferent cells that project to the basilar papilla. No lagenar efferent neurons, however, were found near the superior olivary nucleus where the ventrolateral group of cochlear efferents is located. Whether efferent neurons in the pontine RF send collaterals to both the basilar papilla and to the macula lagena has yet to be determined.

Acoustic Maculae↗
PubMed2005

[Application of methods of heat retrieval of antigens in paraffin sections of rat brain].

The aim of this study was to develop an optimal protocol of processing of rat brain sections permitting to minimize the shortcomings of the method of heat retrieval of antigens and preserving its high efficiency. The research was conducted using the methods of heat antigen retrieval, immunohistochemistry and light microscopy. It was shown that prolonged incubation in blocking solution, containing normal serum of an animal--source of secondary antibodies, eliminated false-positive reaction, demonstrated after heat antigen retrieval in paraffin sections of rat brain.

Animals↗
PubMed1995

Effects of fixation on the preservation of peroxisomal structures for immunofluorescence studies using HepG2 cells as a model system.

The immunofluorescence technique has become an important tool for the investigation of peroxisomes in cell culture. We have used this method for the study of peroxisomes in the human hepatoblastoma cell line HepG2. A marked heterogeneity of peroxisomal forms was detected. Besides spherical (about 100 nm) and rod-shaped structures (about 300 nm) many elongated, undulating tubular forms (up to 5 microns) were found. Further observations indicate that the appearance of the peroxisomal forms in immunofluorescence is dependent on the fixation procedure used. Whereas the fixation with methanol-acetone (-20 degrees C) or ethanol results in a punctate pattern with spherical particles, the use of formaldehyde/Triton X-100 fixation shows well-preserved tubules and rods. These observations may be of special importance for studies on the biogenesis of peroxisomes.

Acetone↗
PubMed1997

Membrane skeletons in avian erythrocytes as revealed by the quick-freezing and deep-etching method.

Ultrastructure of chicken erythrocytes were examined by the quick-freezing and deep-etching (QF-DE) method. Some erythrocytes were fixed with paraformaldehyde and prepared with erythrocyte-splitting method or saponin treatment to remove soluble proteins before quick-freezing. Others were prepared in the cytosol buffer with the erythrocyte-splitting method to obtain natural state of cytoskeletons. Non-expanding membrane skeletons were highly condensed on the cytoplasmic side of lipid membrane in the paraformaldehyde-fixed specimens. Under unilateral extension of the specimens, long stretched filaments were connected alternately with condensed filamentous or granular structures under erythrocyte membranes. As the membrane skeletons got closer to the marginal bands, they become more dense network structures. Moreover, in the fresh unfixed specimens, dense networks of filaments were localized underlying erythrocyte membranes in a relatively intact state. Fine filaments connected the marginal microtubule bands to the cytoplasmic sides of erythrocyte membranes. The different distribution of each cytoskeletal component and the association of these structures may support the elliptocytic shape of chicken erythrocytes and resist the dynamic circumstance.

Animals↗
PubMed1977

[Radial complement fixation test and convex lens method for the determination of antibodies in tissues].

The radial complement fixation test has to be made in forms in 1.4% layer of agar in physiological saline in a Petri dish. These forms have to be of the same thickness as the reaction itself, usually about 1 mm. In the first layer, it is recommended to mix antigen with complement and sheep erythrocytes in agarose and this mixture is to be poured over the tissue fragments examined. Following the primary incubation (at room temperature overnight), another mixture of haemolysin in agarose is poured over the first layer in form of optical convex lenses. The haemolytic phase is performed at room temperature, too. The complement is titrated together with the optimal amount of antigen and a drop of positive serum is placed in the well just in the centre of the first layer. In this way an optimal dilution of complement is detected, which is capable of producing a full hemolysis outside the zone of the positive serum diffused during the first, primary incubation period, whereas the zone itself remains untouched with the haemolytic activity of complement.

Animals↗
PubMed1997

Method for the purification of tissue DNA suitable for PCR after fixation with Bouin's fluid. Uses and limitations in microsatellite typing.

Paraffin-embedded tissues are often the only available material to perform polymerase chain reaction (PCR)-based analysis in various medical purposes. Unfortunately, the use in many countries of acid fixatives such as Bouin's fluid limits the use of such a material for molecular analysis. This article reports the methodological details of a DNA purification technique from Bouin-fixed and paraffin-embedded samples based on a double washing, in an alcohol then in an aqueous medium, of the DNA, which enables PCR reactions from this material. Comparison of the results with those obtained by organic solvent purification of DNA from frozen tissue fragments showed excellent reproducibility in terms of detection of an amplification product on agarose gel. However, differences between the methods were quite frequently seen in the allelic typing profile of microsatellite sequences (CA repeats), either as neo-alleles or by the loss of normal alleles in the fixed materials that constitute a limitation in using DNA from Bouin-fixed tissue as a substrate for fine allelotyping.

Acetic Acid↗
PubMed1978

Long term fixation for histological studies.

It was found that a glutaraldehyde-formalin-phosphate buffered fixative could preserve retinal tissue for light microscopy, even if the tissue remained in the fixative for a month. Retinal tissue that previously was difficult and expensive to procure could now be obtained.

Animals↗
PubMed1993

The effect of formalin fixation on DNA and the extraction of high-molecular-weight DNA from fixed and embedded tissues.

The effect of formalin fixation on DNA and extraction of DNA from fixed tissues was investigated to retrieve archival tissue samples stored in pathology departments for molecular biological studies. Aldehyde fixatives resulted in degradation of DNA at room temperature but not at 4 degrees C. The degradation also occurred in formalin when the pH or the salt concentration was low, or the formic acid level was high. Restriction endonuclease digestion of fixed DNA was incomplete after formalin fixation and this was also temperature-dependent. Thus, relatively intact DNA was obtained from the tissues fixed in buffered formalin at 4 degrees C or fixed with microwave irradiation. The use of modified tissue-lysing buffer containing 4M urea allowed extraction of high-molecular-weight DNA suitable for Southern blot analysis from fixed and embedded tissues. In conclusion, fixation with buffered formalin at 4 degrees C permitted extraction of DNA of sufficient quality for Southern blot analysis.

Aldehydes↗
PubMed1989

The effects of progressive formaldehyde fixation on the preservation of tissue antigens.

A wide variety of fresh post-mortem tissues was fixed in 4% formaldehyde for 6 hours, 1, 3, 7, 14 and 30 days to simulate possible fixation schedules in the diagnostic laboratory, before processing through alcohol and chloroform. The immunoreactivity of paraffin-embedded sections to 25 monoclonal and polyclonal antibodies commonly used in tissue diagnosis was tested employing an avidin-biotin-peroxidase technique. There was a distinct fall-off in the staining of many antigens including the lymphocyte antigens LN1, LN2, LN3 and UCHL1 after 3 days of fixation and the intermediate filament proteins vimentin, desmin and neurofilaments failed to be labelled by monoclonal antibodies after 1 day and only variable staining for the cytokeratins was retained. S100 protein, prostate specific antigen, thyroglobulin and carcinoembryonic antigen were more resilient and showed weak staining after 14 days exposure to formaldehyde. Trypsinization improved the staining for cytokeratins, neurofilaments, desmin and Factor VIII-related protein, but for other antigens, the immunoreaction was weaker and background staining was increased. For the effective application of immunohistochemical staining as a diagnostic tool, the duration of formaldehyde fixation should be kept to a minimum and enzyme digestion should only be judiciously employed for selected antigens.

Adult↗
PubMed1989

Light and electron microscopic demonstration of extracellular immunoglobulin deposition in renal tissue.

Extracellular immunoglobulin (IgG) deposits were shown by both light and electron microscopy in renal biopsy material using immunogold labelling. After fixation of tissue in 4% paraformaldehyde and embedding in Lowicryl K4M, semithin sections were cut and stained using the immunogold silver stain. The sections were then viewed and areas of interest were noted; ultrathin sections were cut from the same block of tissue, then stained using immunogold. Good localisation was achieved at both optical and ultrastructural levels allowing direct correlation to be made in the same area of tissue.

Glomerulonephritis, Membranous↗
PubMed1992

Complete penetration of antibodies into vibratome sections after glutaraldehyde fixation and ethanol treatment: light and electron microscopy for neuropeptides.

To develop a method for quantitative electron microscopic immunocytochemistry on neural tissue of CNS, we tested the extent to which ethanol treatment would improve the penetration of immunoreagents through vibratome sections fixed in high concentrations of glutaraldehyde without compromising ultrastructure. Transverse or sagittal vibratome sections (60-80 microns) of spinal cord perfused with 1% formaldehyde plus 1% or 2.5% glutaraldehyde were washed in 50% ethanol for 0-70 min and stained to reveal immunoreactivity for neuropeptide Y (NPY). Semi-thin (1 micron) or ultra-thin sections were used to assess the depth to which NPY nerve fibers in the dorsal horn were stained. Without ethanol washing, immunoreactive nerve fibers were visualized only in the surface 5-10 microns of transverse or sagittal vibratome sections. In transverse vibratome sections, NPY nerve fibers, which ran perpendicular to the cut surfaces of the sections, were entirely stained after a 30-min wash in 50% ethanol. The numbers of NPY-immunoreactive varicosities and synapses were comparable at the surfaces and in the centers of the vibratome sections. In sagittal sections, where NPY nerve fibers ran parallel to the cut surfaces, fibers in the centers of vibratome sections could not be labeled even after 70 min in 50% ethanol. Substance P- and enkephalin (Enk)-immunoreactive nerve fibers could also be completely stained in transverse sections of spinal cord or medulla oblongata after 30-min exposure to ethanol. Ethanol washing had no significant deleterious effects on ultrastructure, although the amount of cytoplasmic matrix in neurons decreased with increasing exposure. These results indicate that washing with 50% ethanol for at least 30 min allows immunoreagents to penetrate completely through nerve fibers fixed with high concentrations of glutaraldehyde, as long as the fibers have cut ends at both surfaces of a vibratome section. This technique makes possible quantitative electron microscopic immunocytochemical studies and is proving a useful tool for defining synaptic connections in the CNS.

Animals↗
PubMed1993

Microwave fixation for the determination of cerebral blood volume in rats.

The cerebral blood volume (CBV) is sensitive to changing hydrostatic pressures. Thus, measurement methods that rely on removing tissue from unfixed brain may lead to underestimates of the CBV due to the loss of blood from the tissue. In situ fixation of tissue before removal may offer improved accuracy. We employed a triple-label method to measure simultaneously whole brain CBF and CBV in halothane-anesthetized Sprague-Dawley rats, which were then killed either by focused microwave irradiation (approximately 8 kW of incident power x 770 ms) or by decapitation. CBF was measured with [3H]nicotine while the CBV was determined as the sum of the cerebral red cell volume (CRCV--measured with 99mTc-labeled red cells) and the cerebral plasma volume (CPV--measured with [14C]dextran). Animals were studied during hypocarbic (PaCO2 approximately 25 mm Hg), normocarbic, or hypercarbic (PaCO2 approximately 70 mm Hg) conditions. Added studies were performed to verify that the microwave irradiation scheme used was capable of fixing previously administered tracers in place, and also halting the entry of tracer given after irradiation. Results indicate that the method of killing had no effects on CBF measurements, as assessed either by absolute values during normocarbia or responsiveness to changing PaCO2. However, all three volume measurements made using nondiffusible tracers (CRCV, CPV, and CBV) were significantly lower in animals killed by decapitation. Furthermore, CO2 responsiveness for all three variables (as assessed by the slope of the PaCO2/volume) was not evident in decapitated animals. We conclude that in situ fixation offers significant advantages when examining the cerebral distribution space of nondiffusible tracers.

Animals↗
PubMed2005

Initial vascularization and tissue differentiation are influenced by fixation stability.

Fracture healing requires a certain degree of mechanical stability and an adequate blood supply. The hypothesis of the present study was that increased interfragmentary shear leads to a reduced initial vascularization and prolonged healing. The aim of the study was to quantitatively analyze the histological appearance of vascularization and tissue differentiation with regard to fracture stability during the course of healing. A mid-shaft osteotomy of the tibia was performed in two groups of sheep and stabilized with either a rigid or semirigid external fixator, differing in bending stiffness. Interfragmentary movements and ground reaction forces were evaluated in vivo during a 9-week period. The sheep were sacrificed at 2, 3, 6, and 9 weeks postoperatively. The tibiae were tested biomechanically and histological sections from the callus were prepared for analysis of tissue differentiation and vascularization. Larger interfragmentary shear movements in the semirigid fixator group were associated with a reduced initial blood supply. At 6 weeks the semirigid fixator group showed a significantly lower percentage of mineralized bone and a higher amount of fibrous tissue leading to a significantly lower stiffness of the callus than the rigid fixator group. This initial delay in healing was compensated for in the later stages with the production of greater volumes of callus tissue so that both groups showed the same callus stiffness at 9 weeks. However, the rigid fixator group showed signs of the beginning of callus remodeling at the latest time points suggesting a faster bone healing. The results indicate the important role of the initial mechanical stability specifically in the vascularization of an osteosynthesis. Further studies should illustrate the precise role of mechanical conditions on the regulation of angiogenesis during early bone healing.

Animals↗
PubMed1990

Comparison of three silver stains for demonstrating neurofibrillary tangles and neuritic plaques in brain tissue stored for long periods.

Three methods were compared to find a reliable method for demonstrating neurofibrillary tangles (NFTs) and neuritic plaques (NPs) in brain tissue stored for long periods in formalin or as paraffin blocks. The short-term fixation of tissue, e.g. up to 6 months in formalin does not usually present a problem using any of the three methods tried, e.g. Gallyas, modified Palmgren, or modified Bielschowsky, but once the time lengthens to 6 years or more the demonstration of NFTs and NPs is not so reliable using the first two methods. The modified Bielschowsky method, however, demonstrates well both NFTs and NPs in material stored in formalin or as paraffin blocks for long periods, e.g. 7 years, and also compares favourably with the other methods on freshly processed material and fixed tissue, stored for shorter periods. We also noted as a consistent trend, irrespective of the staining technique employed, the detection of fewer plaques and tangles in material stored in formalin, as opposed to that stored as blocks embeded in paraffin wax.

Aged↗