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PubMed2005

A quantitative comparison of synaptic density following perfusion versus immersion fixation in the rat cerebral cortex.

The main objective of the present study was to perform an unbiased comparison of immersion vs. perfusion techniques to assess whether we could use the former to quantify synapses through electron microscopy (EM). Using the immersion technique is ideally suited for instances in which the specimen under study could not be perfused under the standard EM protocol. Our results suggest that, despite suboptimal qualitative results, fixation by immersion allows for adequate quantification of synapses.

Animals↗
PubMed2005

Comparison of F-actin fluorescent labeling methods in pollen tubes of Lilium davidii.

Fluorescence labeling of F-actin in pollen tubes by various methods has produced inconsistent results in the literature. Here, we report that EGTA, which was always used in fixative buffers in the past and thought to help cytoskeleton stabilization, can significantly affect F-actin distribution and lead to the formation of thick F-actin bundles at the tip of the pollen tube. We also found that vacuum-infiltration for the first 5 min during pollen tube fixation can better preserve normal cytoplasm structure and F-actin distribution. In contrast, m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) treatment before chemical fixation resulted in a shortening of the free zone of thick F-actin bundles in the pollen tube tip. Taken together, our results suggest that exclusion of EGTA and MBS from the fixative buffer, in combination with vacuum-infiltration in the first 5 min of fixation, can improve F-actin fluorescence labeling in pollen tubes of Lilium davidii.

Actins↗
PubMed1997

Microwave fixation of rabies specimens for fluorescent antibody testing.

An adaptation of the conventional procedure for fixation of brain impressions prior to rabies testing was developed using a microwave oven to fix slides in less than 1 min. A total of 2394 routine specimens were examined to evaluate the efficacy of microwave fixation of brain impressions as an aid to shortening the turnaround time for reporting. Slides were fixed for 55-60 s in a standard household microwave oven at 50%, power using phosphate buffered saline supplemented with 3% Tween 20 as a fixative medium. They were then stained using the standard rabies immunofluorescent staining procedure with minor modifications. This process potentially could allow results to be reported to submitters within 1.5 h after specimens arrive in the laboratory. Microwave fixation was in complete agreement with conventional acetone fixation for detection of positive rabies specimens.

Animals↗
PubMed2000

Influence of different fixation procedures on the quantification of infarction and oedema in a rat model of stroke.

In pharmacodynamic studies using focal ischaemia models, the size of the infarct measured by quantitative histology is the most important outcome measure. Precise, unbiased and reproducible assessment of infarct volume is of foremost importance. A frequent problem in interventional stroke models is the evaluation of infarcts in animals found dead, where instant post-mortem fixation of the brain cannot be performed. The purpose of this study was to investigate possible bias from perfusion, immediate and 3-h post-mortem delayed immersion fixation on the measured volumes of cerebral infarction, oedema and hemispheres in a rat embolic stroke model. Thirty-six male Sprague-Dawley rats were thromboembolized into the internal carotid artery. After survival for 24 h, the animals were divided into three groups: group 1 - immediate perfusion fixation; group 2 - immediate immersion fixation of the brain; and group 3 - animals left dead for 3 h at room temperature before removal of the brain for immersion fixation. Following histological preparation and evaluation, the volumes of the hemispheres and infarction were measured by quantitative histology and planimetry. Brains fixed by immersion were 7% larger than the perfusion-fixed brains. Delaying the immersion fixation for 3 h may increase hemisphere volume by a further 12%. Independent of the fixation procedure, the size of infarction was approximately 40% of the ipsilateral hemisphere, and the oedema was approximately 11% of the size of the infarct. The used planimetric technique was accurate with measured values within +/- 2% of the factual value. In conclusion, sizes of hemispheres, infarction and oedema in absolute volume measures are influenced by the effect of unwanted variation of brain size caused by biological factors and artificial shrinkage caused by fixation, dehydration and heat treatment of the specimens. Infarction and oedema expressed relatively in per cent of hemisphere and infarct, respectively, are robust measures independent of the investigated fixation procedures.

Animals↗
PubMed1993

NADPH-diaphorase (NOS) is induced in pyramidal neurones of hippocampal slices.

We found NADPH-diaphorase (presumably identical with nitric oxide synthase) in pyramidal neurones of the hippocampus in slices that stayed in a chamber for 30 min or longer. In some instances parallel slices showed normal membrane properties when studied electrophysiologically. In freshly made slices the pyramidal neurones were not stained. Thus, after induction of the enzyme, the hippocampal pyramidal neurones can synthesize nitric oxide which may serve as a retrograde messenger in long-term potentiation. The enzyme may also play a role in cell loss seen in slices which stayed in a chamber for 9-22 h before fixation.

Animals↗
PubMed1998

5'-nucleotidase in rat photoreceptor cells and pigment epithelial cells processed by rapid-freezing enzyme cytochemistry.

This report describes the subcellular distribution of 5'-nucleotidase (5'-NT) in rat photoreceptor cells and pigment epithelial cells processed by rapid-freeze enzyme cytochemistry. There was a striking difference in the ultrastructural localization of 5'-NT activity between rod outer segments after freeze-substitution fixation and conventional fixation. By rapid-freezing enzyme cytochemistry, 5'-NT activity was localized in the extradiscal space of intact nonvacuolated discs, whereas by conventional cytochemistry it was shown in the intradiscal space of artifactual vacuolated discs. In the freeze-substituted retinal cells, an appreciable difference in functional 5'-NT molecules was also found. The soluble 5'-NT on the cytoplasmic side of the disc membrane was vital in the rod outer segments, whereas the membrane-bound ecto-5'-NT on the exoplasmic (external) surface of the apical process was active in the pigment epithelial cells. Rapid-freezing enzyme cytochemistry should be worth employing as a method to reveal the fine localization of enzyme activity at the level of cell ultrastructures, which are poorly preserved by conventional fixation, and should provide information approximate to that in living cells.

5'-Nucleotidase↗
PubMed1996

[Optimal fixation for the detection of anti-neuronal antibody by immunohistochemistry in the paraneoplastic syndrome].

Paraneoplastic syndrome(PS) associated anti-neuronal autoantibodies are characterized by their antigen molecular weight determined by Western blot and immunostaining pattern recognized through immunohistochemistry. We investigated immunohistochemical fixatives for their sensitivity in the detection of anti-neuronal nuclear antibody(Hu). Serum used for this study was taken from a patient with anti-Hu antibody seropositivity, which was ascertained by recombinant Hu protein. Western blot analysis produced 37kDa band. We examined six fixative conditions: immersion fixed with acetone, Bouin's solution, Sakura rapid fixative("Ufix'), and perfusion fixed with 2%, 4%, 8% paraformaldehyde (PFA) on the basis of each immunoreactivity in a rat cerebellum, brain stem and liver. The optimal fixation for detecting anti-Hu antibody was perfusion fixed with 2% PFA, that reacted conspicuously with nucleus but not nucleolus of neurons. The perfusion method proved superior to immersion in immunostaining intensity. With immersion fixation, specific immunostaining pattern lessened and differentiation from cytoplasm decreased. With various concentrations of PFA, immuno-reactivity with nucleus at 2% PFA was similar to 4%, although serum optimal dilution at 2% was slightly greater than 4%. The variety of staining patterns of anti-Hu antibody is closely related to the vulnerability of neuronal antigens to the fixatives. The detection of anti-neuronal antibodies is important for early diagnosis and treatment of occult tumors. The immunostaining method is a useful and sensitive way to research these antibodies. Therefore, it is essential to consider the selection of fixation and the preservation of the antigenicity in evaluating immunohistochemical hallmarks.

Animals↗
PubMed2004

Influence of lung fixation technique on the state of alveolar expansion-a histomorphometrical study.

Lungs removed at necropsy normally collapse due to the loss of negative pleural pressure leading to a quite unnatural appearance of both gross and histological specimens. In order to demonstrate the influence of post-mortem lung retraction on the degree of alveolar expansion, a histomorphometrical analysis was performed in lungs from a 9-month-old healthy infant. Tissue specimens from the right lung were obtained at autopsy and routinely fixed after retraction ('routinely fixed lung'), whereas the left lung was fixed in situ before opening the thoracic cage ('in situ fixed lung'). The size of the alveoli as well as the thickness of the alveolar walls were measured using an automatic image processing and analysis system (Leica QWIN) in both lungs. The mean alveolar size was 8.7 x 10(3) microm(2) in the routinely fixed lung (alveoli, n=1.1576) and 10.9 x 10(3) microm(2) in the in situ fixed lung (alveoli, n = 841). In contrast, the diameter of the alveolar walls showed no significant difference in both lungs. The average thickness of the alveolar walls was 7.9 microm (measuring sites, n = 1.190) in the routinely fixed lung and 8.1 microm in the in situ fixed lung (measuring sites, n = 1.027), respectively. The results provide evidence of significantly reduced aeration in the retracted and routinely fixed lung which could be of special forensic interest in cases of suspicious infanticide, stillbirth or infant death by drowning or suffocation.

Forensic Medicine↗
PubMed1995

The arrangements of F-actin, tubulin and fodrin in the organ of Corti of the horseshoe bat (Rhinolophus rouxi) and the gerbil (Meriones unguiculatus).

The composition of cytoskeletal elements in hair cells and non-sensory cells was studied in paraformaldehyde fixed cochleae of the horseshoe bat and the gerbil using phallotoxins and antibodies directed against actin, alpha-tubulin and fodrin. In both species, cryostat sections of the organ of Corti were studied using confocal fluorescence microscopy; in the bat, ultrathin sections were investigated using actin-immunoelectron and classical electron microscopy. F-actin was found in stereocilia and cuticular plates of inner and outer hair cells (IHCs and OHCs) of both species. In fixed material from both species, no F-actin staining was detected in the cytoplasm or along the lateral cell membrane of OHCs, whereas in freshly isolated OHCs of the gerbil, a faint F-actin staining was detected along the lateral wall. In the bat, the patterns of F-actin staining were confirmed with actin-immunoelectron microscopy. The alpha-tubulin antibody strongly labeled IHCs of both species. They contained a complex network of microtubules especially in the neck portion. In the bat, OHCs showed no distinct alpha-tubulin reactivity, as would be expected given the scarcity of microtubules observed at the ultrastructural level. In the gerbil, alpha-tubulin reactivity was found throughout the OHC body with highest intensity in the cell apex. In Deiters cells, pillar cells and Boettcher cells of both species, F-actin and microtubules were colocalized at contact zones with the basilar membrane. In Deiters cups, F-actin staining was most pronounced in the basal turn of the bat cochlea. In the gerbil, a distinct baso-apical gradient was found in immunostaining properties and morphology of the Deiters cells. Intense fodrin reactivity was found in the cuticular plates and along the lateral cell membrane of both types of hair cells of the bat. Cytoplasmic fodrin staining was localized within the IHCs of the bat. In the gerbil, intense fodrin staining was only found in cuticular plates of hair cells and staining of the lateral cell membrane of hair cells was faint. A faint fodrin staining was also seen in Deiters cells of both species. The basic arrangement of the cytoskeletal elements in the batś organ of Corti is similar to that of other mammals, however, certain features suggest the presence of subtle differences in micromechanical properties: there is an increased concentration of microtubules in the neck portion of IHCs, an increase in the amount of F-actin within the Deiters cups and a reduced amount of microtubules in the OHCs.

Actins↗
PubMed2000

Fixation of transsynaptically transported WGA-HRP and fluorescent dyes used in combination.

The transsynaptic transport of WGA-HRP is a powerful tool for the identification of last order interneurones in the spinal cord, but differentiation of primarily labelled motoneurones from transsynaptically labelled interneurones can be difficult. We therefore combined the transsynaptic labelling of interneurones with WGA-HRP and the labelling of motoneurones with fluorescent dyes (fluorogold, red beads, fast blue, DAPI) by injecting a mixture of the tracers into the nervi mediani of rats. The influence of different fixatives on the simultaneous preservation of WGA-HRP and fluorescence is evaluated. It is shown that, of several perfusion protocols, only perfusion with paraformaldehyde in addition to 1.4% lysine and 0.23% periodate allowed simultaneous visualisation of transsynaptic WGA-HRP and fluorescent dyes.

Acetaldehyde↗
PubMed1996

Microwave-enhanced fixation of rabbit articular cartilage.

Cartilage, being highly aqueous, is difficult to preserve for electron microscopy without artefacts. Microwave-enhanced fixation is suggested as a standard method for block samples of this material, with dimensions of up to 12 x 7 x 3 mm. Cartilage samples from the tibial plateau of adult rabbits were fixed by conventional, cryo- or microwave-enhanced fixation. Constant or cyclical microwave irradiation of samples, immersed in fixatives, was carried out to varying final solution temperatures. Microwave-enhanced fixation and staining is shown to be both rapid and reproducible, giving fine structural preservation. Below 323 K microwave fixation always gave excellent preservation of the fine structure within seconds. At higher temperatures thermal artefacts were introduced. In this study the microwave-enhanced fixation is equal in quality to the test conventional immersion fixation and is nearly as fast as cryo-preservation. It provides a standardized, reproducible fixation for morphological studies on cartilage with good process control.

Animals↗
PubMed1991

Distinction between antinuclear antibody and P-ANCA.

To differentiate between perinuclear immunofluorescence staining of antinuclear antibody (ANA) and the perinuclear form of anti-neutrophil cytoplasmic antibody (P-ANCA), the pattern after formaldehyde vapour fixation of normal human neutrophils was compared with that of standard ethanol fixation. Fifteen out of 17 myeloperoxidase antibody positive sera showed cytoplasmic staining on formaldehyde vapour fixed cells; 30 of the 32 ANA positive samples became negative or gave weak nuclear staining on the same substrate. Formaldehyde vapour fixation is a simple, useful technique for differentiating between ANA and P-ANCA.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1992

Assessment of specimen fixation in a surgical pathology service.

The quality of specimen fixation was examined within a routine diagnostic histopathology service. For each specimen the adequacy of fixation was assessed and the transit time between operating theatre and the laboratory was measured. Preliminary fixation was found to be inadequate in 25% of specimens and some form of manipulation to assist fixation was required in 36% of specimens. The mean transit time was 22 (SD 10.7) hours. Specimen fixation and transport are additional factors to consider in quality assurance of histopathology.

England↗
PubMed1991

The regulation of rat brain pyruvate kinase by estradiol-17 beta.

The effect of estradiol-17 beta on the activity of pyruvate kinase from rat brain was investigated at initial stages of hormone administration. After estradiol treatment the rise of pyruvate kinase activity was found in the firmly bound synaptosomal fraction, whereas pyruvate kinase activity in soluble and weakly bound fractions has been reduced. The activation of the enzyme was also discovered after glutaraldehyde fixation on synaptosomal membranes. The possible mechanism of extragenomic estradiol action is discussed.

Animals↗
PubMed1993

Microwave-assisted staining of mucosal mast cells and granulated intra-epithelial lymphocytes after formalin fixation.

Rat jejunum was fixed with either formalin or methanol-formalin acetic acid (MFAA) and stained with Astra Blue or Alcian Blue with or without microwave irradiation. Staining of both mucosal mast cells and granulated intra-epithelial lymphocytes after formalin fixation was considerably improved by microwave irradiation. On the other hand, microwave irradiation slightly impaired staining of mucosal mast cells (MMC) and even more strongly granulated intra-epithelial lymphocytes (GIEL) after MFAA fixation.

Acetates↗
PubMed1997

Number, fixation properties, dye-binding and protease expression of duodenal mast cells: comparisons between healthy subjects and patients with gastritis or Crohn's disease.

There is an accumulation of evidence to suggest that mast cells may play a key role in gastrointestinal inflammation. We have investigated the numbers and heterogeneity in staining properties of mast cells in biopsies of the duodenum of normal subjects (n = 10), and of normal duodenum from patients with Crohn's disease of the ileum and/or colon (n = 7) or with Helicobacter-associated gastritis of the antrum/corpus (n = 6). In normal donors, two subsets of mast cells, one located in the duodenal mucosa and the other in the submucosa, were clearly distinguished by their morphology and dye-binding properties. Whereas submucosal mast cells stained metachromatically with Toluidine Blue after neutral formalin fixation and emitted a yellow fluorescence after staining with Berberine sulphate, those in the mucosa were invisible using these stains. In patients with gastritis or Crohn's disease, there were marked changes in the numbers of mucosal mast cells compared with control subjects even though the duodenal biopsies were from apparently uninvolved tissue. Gastritis was associated with increased mucosal mast cell numbers (controls: 187 +/- 23 cells mm-2; gastritis: 413 +/- 139 cells mm-2; p = 0.0004), but mean mucosal mast cell counts in the uninvolved duodenum of Crohn's patients were actually decreased (34 +/- 30 cells mm-2, p = 0.0147). The clear differentiation between mucosal and submucosal mast cells on the basis of metachromasia with Toluidine Blue was not seen in biopsies from the patients with gastritis or Crohn's disease. Previous studies which have suggested that there are no distinct mucosal and submucosal mast cell subsets in the human intestine may, therefore, have been affected by the use of tissue from diseased subjects. Heterogeneity in the expression of mast cell tryptase and chymase was seen by immunohistochemistry using specific antibodies, but the relative numbers of mast cell subsets were critically dependent on the methods used. Using a sensitive staining procedure, the majority of mucosal mast cells stained positively for chymase as well as for tryptase, an observation confirmed by immunoelectron microscopy and immunoabsorption studies. Our findings suggest that early stages in intestinal inflammation may be reflected in changes in mast cell numbers and in their staining properties, and call for a reappraisal of mast cell heterogeneity in the human intestinal tract.

Adult↗
PubMed1990

Detection of JC virus DNA by polymerase chain reaction in patients with progressive multifocal leukoencephalopathy.

Polymerase chain reaction (PCR) was used in the detection of JC virus (JCV) DNA in formalin-fixed, paraffin-embedded brain tissue sections from 24 patients with progressive multifocal leukoencephalopathy. Brain sections from normal autopsies (n = 17) and other neurologic conditions (n = 4) were used as controls. Specific amplified products were detected in 20 (83%) of 24 patients with progressive multifocal leukoencephalopathy. PCR did not amplify JCV or human beta-globin gene sequences in four patients with characteristic demyelinating lesions of progressive multifocal leukoencephalopathy that were positive for JCV by in situ hybridization or immunocytochemistry. PCR from biopsy sections resulted in more intense amplification signals than PCR from autopsy tissue. Normal brain sections from 17 autopsies were negative by PCR. Low-grade amplification of JCV was observed in one patient with herpes simplex virus encephalitis. PCR served as a more rapid test for confirmation of progressive multifocal leukoencephalopathy than in situ hybridization. However, PCR performance was altered by prolonged formalin fixation of tissue and undetermined inhibitors of the amplification reaction. Laboratories and clinicians should be aware of the difficulties encountered when using paraffin-embedded tissue for PCR.

Base Sequence↗