Fixation Artifacts Research Articles

Interspecies variations in oral epithelial cytokeratin expression.

The aim of this study was to determine the degree to which the epidermis and oral epithelium of species other than man express cytokeratin (CK) intermediate filaments, which are markers of epithelial differentiation. Fixed, wax-embedded samples of skin, buccal mucosa and gingiva from rhesus monkey, marmoset, cow, sheep, pig, ferret, hamster, axolotl and trout were tested for CK expression using a panel of antihuman CK antibodies and an immunoperoxidase procedure. Human skin and oral mucosa were also stained to act as positive control. The results showed that antihuman CK antibodies stained animal tissues, but the patterns of staining were not always identical to the established human CK profile. Of particular interest was the expression of CK18, typically only detected in 'simple' epithelium in man, in bovine, ferret and hamster stratified epithelium from different sites. However, there was evidence of variable anti-CK antibody cross-reactivity, both as a result of intrinsic variations in CK polypeptide structure and as artifacts of fixation. We conclude that some CK are conserved between species, but that biological variables, for example local functional requirements, and technical factors affect the results. These considerations need to be borne in mind in animal studies of epithelial differentiation employing CK immunohistochemistry. Biochemical characterisation is ultimately necessary to determine specific differences between human and animal CK.

One hundred years of Golgi's "perineuronal net": history of a denied structure.

Perineuronal nets are reticular structures enwrapping cell bodies and the largest dendrites of several neuronal populations. Discovered by Camillo Golgi, who described them in detail in 1898, they were intensely studied by the most famous contemporary neurohistologists for about twenty years. The opinion of Ramon y Cajal that perineuronal nets were a fixation artifact ended the first period of studies. Only a few researchers, among whom the Italian neurologists Besta and Belloni, went on with their studies up to the 1930s documenting the morphology of perineuronal nets of different mammals and of man both in normal and in pathological conditions. Only after about fifty years, the advances in the field of cytochemistry allowed the elucidation of not only the actual existence of perineuronal nets, but also their chemical nature, showing conclusively that they are complex organisations of extracellular matrix molecules, namely glycoproteins and proteoglycans. The research on perineuronal nets today involves several groups engaged to elucidate their biological properties and functional role.

Dynamic Rearrangement of the Filamentous Actin Network Occurs during Zoosporogenesis and Encystment in the OomycetePhytophthora cinnamomi

The organization of filamentous actin (F-actin) in living cells of the oomycetePhytophthora cinnamomiwas determined during zoosporogenesis and zoospore encystment by microinjecting sporangia with fluorescently labeled phalloidin and observing resultant fluorescence by confocal microscopy. In multinucleate sporangia prior to the induction of cleavage, phalloidin labeling took the form of plaques which occurred mainly in the periphery of the sporangia. After induction of cleavage, phalloidin labeling showed that the plaques disappeared and that F-actin began to accumulate along the developing cleavage planes and around nuclei and water expulsion vacuoles. F-actin labeling was also observed near the plasma membrane in zoospores and young cysts but reverted to the plaque form in older cysts. Localization of F-actin close to the developing cleavage planes is consistent with the idea that actin microfilaments function in the positioning and expansion of the cleavage membranes. Observations of plaques of actin in living sporangia provide evidence that plaques are not aldehyde-induced fixation artifacts.

Electron tomography of mitochondria from brown adipocytes reveals crista junctions.

Electron microscope tomography was used to examine the membrane topology of brown adipose tissue (BAT) mitochondria prepared by cryofixation or chemical fixation techniques. These mitochondria contain an uncoupling protein which results in the conversion of energy from electron transport into heat. The three-dimensional reconstructions of BAT mitochondria provided a view of the inner mitochondrial membrane different in important features from descriptions found in the literature. The work reported here provides new insight into BAT mitochondria architecture by identifying crista junctions, including multiple junctions connecting a crista to the same side of the inner boundary membrane, in a class of mitochondria that have no tubular cristae, but only lamellar cristae. Crista junctions were defined previously as the tubular membranes of relatively uniform diameter that connect a crista membrane with the inner boundary membrane. We have also found that the cristae architecture of cryofixed mitochondria, including crista junctions, is similar to that found in chemically fixed mitochondria, suggesting that this architecture is not a fixation artifact. The stacks of lamellar cristae extended through more of the BAT mitochondrial volume than did the cristae we observed in neuronal mitochondria. Hence, the inner membrane surface area was larger in the former. In chemically fixed mitochondria, contact sites were easily visualized because the outer and inner boundary membranes were separated by an 8 nm space. However, in cryofixed mitochondria almost all the outer membrane was observed to be in close contact with the inner boundary membrane.

DETECTION OF PARVOVIRUS B19 INFECTION IN FIRST AND SECOND TRIMESTER FETAL LOSS

Fetal and placental tissues and maternal sera from a series of 273 cases of first and second trimester fetal loss were collected to detect the frequency of parvovirus B19 infection. In addition, fetal tissues were studied for the presence of congenital anomalies. Serology of maternal sera, histology of fetal tissues and placenta, polymerase chain reaction (PCR), in situ hybridization (ISH), and immunohistochemistry (IHC) were used for the detection of parvovirus B19 infection. Sera were tested for B19-specific immunoglobulin M (IgM) and/or IgG using an enzyme-linked immunosorbent assay technique. Based on serology, 149 cases not related to B19 infection were excluded from further analysis. Two of the remaining 124 cases (0.7% of all 273 cases) had parvovirus B19-specific IgM and IgG at the time of abortion, indicating a recent maternal parvovirus B19 infection. In our histological examination, 10 cases contained nuclear vacuolization in fetal erythroid progenitor cells, either in fetal tissues (n = 2) or in placental tissue (n = 8). However, this vacuolization was considered a fixation artifact and not identical to parvovirus B19-specific nuclear inclusions described in previous reports. Only 1 of these 10 cases had parvovirus B19 DNA detectable in placental tissue by PCR analysis. Neither in this case nor in any of the other cases tested was parvovirus B19 DNA or protein detectable by ISH or IHC, respectively. In none of 41 cases in which fetal tissues were available were congenital anomalies found. In conclusion, the frequency of maternal parvovirus B19 infection in this series of fetal losses is low (0.8%). This low frequency does not allow any conclusions with regard to the occurrence of congenital anomalies resulting from parvovirus B19 infection and the usage of nuclear histology for the detection of fetal parvovirus B19 infection is considered a nonspecific parameter that requires confirmation by PCR.

Chapter 9: GFP Variants for Multispectral Imaging of Living Cells

Unlike enzyme markers, green fluorescent protein can be visualized at high resolution in living cells using confocal microscopy. The images are not prone to fixation or staining artifacts, and can be of exceptional clarity. Moreover, the activities of living cells, such as cytoplasmic streaming, are clearly evident during microscopy. Ordinarily, movement within a sample is a nuisance, placing constraints on the use of sometimes lengthy techniques for noise reduction during confocal microscopy, such as frame averaging. However, it is possible to monitor dynamic events by time-lapse confocal microscopy, and this combination of a vital fluorescent reporter with high-resolution optical techniques shows much promise for use in cell biological and physiological experiments. Genetic systems such as that of Arabidopsis provide a large resource of potentially informative mutants, and there has been much recent improvement in techniques for determining the molecular basis of a particular phenotype. The use of fluorescent proteins will provide further tools for examining the biology of mutant cells. The precision with which particular cellular structures can be decorated with GFP and the ease with which subcellular traffic can be monitored indicate that this approach will be very useful for cell biological and physiological observations, particularly for detailed examination of plant mutant phenotypes.

Chapter 8: GFP Fusion Proteins as Probes for Cytology in Fission Yeast

This chapter discusses many practical considerations for expressing green fluorescent protein (GFP) fusion proteins in fission yeast, with an emphasis on guidelines and examples rather than on specific rules or recipes. There are primarily two areas in which GFP fusion proteins may be particularly useful in fission yeast. First, new types of experiments, such as determining protein localization in cells without the potential artifacts of fixation or filming protein localization and dynamics in living yeast cells, have now become possible. In addition, using GFP fusion proteins rather than more conventional methods for seeing proteins inside cells may make specific techniques, such as visual screens for protein localization, much more simple and rapid in execution. Expressed GFP fusion proteins generally need to be visible to the naked eye under the microscope, although if sensitive imaging equipment is available, the expression only just at the limit of detection by the eye may be sufficient. Producing a visible protein may often require higher than normal expression levels and can be achieved by using heterologous promoters, multicopy plasmids, or both. In addition, GFP mutants can now be used in place of the wild-type GFP.

Schizogenous Secretory Cavities of Citrus limon (L.) Burm. F. and A Reevaluation of the Lysigenous Gland Concept

The lysigenous appearance of Citrus limon (L) Burm. f. secretory cavities is the result of fixation artifacts. The glandular epithelial cells of lemon glands undergo rapid destructive swelling when immersed in commonly used fixatives. The swelling is most evident with mature glands, causing a false impression that the epithelial cells undergo autolysis as they complete their development. Epithelial cells of secretory cavities sliced open during tissue preparation show more extensive swelling than those left intact during fixation, indicating a possible cause for inconsistencies among reports of Citrus gland development. Aqueous primary fixation of intact secretory cavities results in sufficient epithelial cell swelling to give a false impression of precocious senescence; osmium vapor-fixed glandular cells show less swelling, contain intact organelles, and appear living at gland maturity. Although the concept of lysigeny is very old, recent evidence for lysigeny is based on investigations that used standard aqueous fixatives. Since fixation requirements for glands from other species of plants could be similar to those for Citrus glands, we suggest that lysigeny, in general, could be a false category of gland development, representing misinterpretation of artifacts.

Comparative evaluation of unfixed and fixed human neutrophils for determination of antineutrophil cytoplasmic antibodies by indirect immunofluorescence.

BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCAs) are found in the sera of patients with vasculitides and ulcerative colitis. Using indirect immunofluorescence on ethanol fixed neutrophils, ANCAs can be divided into two...

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.

The Significance of Central Pathology Review in Clinical Studies of Transitional Cell Carcinoma in Situ

The causes of interobserver variation in the pathological diagnosis of urothelial neoplasia were studied. A central review was performed on pathological specimens in a multi-institutional clinical study of patients with in situ transitional cell carcinoma of the bladder. A significant discrepancy in pathological diagnosis was noted between the original report and the central review in 60 of 159 biopsies (38%) and in 73 of 217 cytology specimens (34%). Biopsy discrepancies were almost equally divided between upgrades and downgrades, whereas 89% of cytology discrepancies involved an upgrade in diagnosis by the central reviewer. The most significant factor causing variability in biopsy diagnoses was the multiplicity of classifications used by the originating pathologists. Other factors included fixation and biopsy artifacts. Cell degeneration secondary to treatment was the most important factor resulting in cytology under grading. At originating institutions the correlation of diagnoses between concurrent biopsy and cytology specimens was poor. The lack of a well accepted standard for the histopathological diagnosis of transitional cell carcinoma in situ poses a major problem for multi-institutional studies of this disease. Organizers must include a histopathological standard in the study plan and publicize it to all participants, particularly pathologists. Central review of pathological specimens is essential to maintain data integrity.

Significance of demonstrable vascular space invasion for the progression of prostatic adenocarcinoma.

Histologically demonstrable vascular invasion by tumor has been reported as an index of poor prognosis correlating with increased probability of metastasis in many types of cancer other than prostatic. We quantitated vascular invasion foci in 357 radical prostatectomy specimens and developed improved criteria for their diagnosis and their distinction from fixation artifact. Vascular invasion foci were found in 7% of cancers less than 4 cc in volume and 24% of larger cancers. Most foci were selectively located either near the basal end of the cancer or near the transition zone border. Correlations among multiple morphologic variables showed significant correlation of vascular invasion only with the presence of intraductal carcinoma. The only statistically significant independent predictors of disease progression (serum prostate-specific antigen elevation) were vascular invasion, carcinoma grade, and cancer volume. Our findings suggest that further study of vascular invasion foci may disclose additional information about the biologic features of local and distant spread of prostatic carcinoma.

Correlation between p53 immunostaining patterns and gene sequence mutations in breast carcinoma.

We performed p53 immunostaining in 82 invasive breast carcinomas by using two commercially available antibodies, one of which (DO7) was employed in formalin-fixed paraffin-embedded sections. The other antibody (PAb1801) was evaluated in corresponding acetone-fixed cryostat sections. A greater percent of cases were immunostained with DO7 compared to PAb1801 (52% vs 33%); however, the staining was more often heterogeneous (6-50% cells positive) or focal (< or = 5% cells positive) with DO7 (9% vs 31%). To investigate the genetic relevance of p53 immunostaining, single-strand conformational polymorphism (SSCP) analysis and DNA sequencing were performed on exons 2-11 by using archival tissue samples of 18 cases that were selected on the basis of certain immunostaining patterns. Two (33%) of six tumors with negative staining for DO7 had gene sequence mutations; however, one of these mutations was a base-pair deletion that caused a reading-frame shift and the other was a base-pair insertion that resulted in a stop codon. Both of these tumors exhibited immunostaining with PAb1801, although it was weak and cytoplasmic in one case. Conversely, three (30%) of 10 tumors showing immunoreactivity in 6-100% of cells with both reagents lacked a gene sequence mutation. Of the remaining seven tumors that were positive by SSCP, six contained a point mutation resulting in a base-pair substitution. Despite repeat analyses, one of the cases positive by SSCP failed to demonstrate a mutation in the sequenced exons. Four (80%) of five cases with heterogeneous DO7 immunoreactivity (that is, 6-50% of nuclei positive) were positive for gene sequence mutation. Neither of two cases showing focal DO7 nuclear staining in < 5% of tumor cells contained a mutation in the sequenced exons, and neither of these cases was strongly positive with PAb1801. Staining for either antibody was significantly associated with adverse outcome, as determined by disease recurrence at 52 months median follow-up (DO7, p = 0.01; and PAb1801 p = 0.002, chi-squared test). We conclude that a variety of factors may account for discrepancies when immunohistology is used to evaluate p53 status. These include fixation artifacts, differing epitope specificities of monoclonal reagents, presence of immunohistologically "silent" mutations and, possibly, aberrant overexpression of wild-type protein.

Conformational changes of smooth endoplasmic reticulum induced by brief anoxia in rat Purkinje cells.

Morphological changes of the smooth endoplasmic reticulum (SER) in rat cerebellar Purkinje cell dendrites were examined under apneic conditions for 1-5 minutes, induced by an incision of the diaphragm and the collapse of the lungs. The dendrites obtained from control rats contained a tubular network of the SER and hypolemmal cisterns adjacent to the plasma membrane. After a 3-5-minute apnea, the cytoplasm was occupied by many flattened cisterns stacked into lamellae, referred to as "lamellar bodies." A quantitative analysis revealed that the number of lamellar bodies became maximum after 3 minutes of apnea. After the treatment time, they increased in size by adding new cisterns to the previous core lamellae. This analysis also showed that the total amount of the SER membranes contained in a dendrite did not change during anoxia. Conformational changes from the tubular or hypolemmal SER to lamellar bodies during brief anoxia might occur through a transient and intermediate form of "fenestrated cisterns," flat across the transverse plane and penetrated by many longitudinally arranged microtubules. We suggest that these morphological changes of the SER during brief anoxia are not fixation artifacts but represent a biological reaction for protecting against intracellular abnormalities during anoxia.

In vivo ANA is a fixation artifact: nucleosome-complexed antinucleosome autoantibodies bind to the cell surface and are internalized.

It has been suggested that binding of anti-double-standed DNA antibodies to cell surfaces, followed by internalization and nuclear binding (so called in vivo ANA) is of pathophysiological significance for tissue damage in systemic lupus erythematosus. We have shown before that pathogenic antinuclear antibodies complexed to nucleosomal antigens can bind to heparan sulfate in the glomerular basement membrane in vivo. Because nucleosomes are also reported to bind to the cell surface, we hypothesized that in vivo ANA is a property of antinuclear antibodies bound to nucleosomal antigens. Therefore, we studied three antinucleosome monoclonal antibodies (mAb) that exhibit in vivo ANA as seen by immunofluorescence in mice inoculated intraperitoneally with the hybridoma producing the mAb. The same mAb complexed to nucleosomal antigens after intravenous injection into mice induced in vivo ANA, in contrast to purified noncomplexed mAb. To study this in more detail, we incubated complexed mAb with various cell lines and found binding to the cell surface and subsequent internalization into cytoplasmic vesicles. However, no binding to the nucleus was observed by immunoelectron microscopy (IEM) and confocal laser microscopy. Noncomplexed mAb did not bind to the cell surface. Next, from mice bearing the hybridomas producing the mAb intraperitoneally, a small part of the kidney was snap frozen in liquid N2, fixed with acetone, and studied in immunofluorescence, whereas the remaining part of the kidney was fixed in vivo by renal perfusion with a mixture of 0.01 M sodium periodate, 0.075 M lysine HCl, 0.0375 M Na2HPO4, and 2% paraformaldehyde (PLP) and studied in both immunofluorescence and IEM. In the acetone-fixed kidney sections obtained without in vivo fixation we again observed in vivo ANA. However, after in vivo PLP perfusion fixation, no nuclear binding was found. In IEM, localization in cytoplasmic vesicles was seen. In conclusion, antinucleosome antibodies complexed to nucleosomal antigens can bind to the cell surface and are transported into the cytoplasm, but do not bind to the nucleus. The reported nuclear localization of antinuclear antibodies is caused by a fixation artifact.

Das hyalinisierende hellzellige Karzinom der Speicheldr�sen

Many cell types of the salivary glands have clear cytoplasm. Causes of clear cytoplasmic quality in light microscopy are loss of organelles, storage of substances or fixation artefacts. Differential diagnosis of the different clear cell types requires special staining techniques, immunocytochemistry and electron microscopy. A new and distinct salivary gland neoplasm is hyalinizing clear cell carcinoma, which was not included in the second edition of the WHO Classification of Salivary Gland Tumors. Analysis of the collected cases of the Salivary Gland Register Hamburg and recent reports in the literature reveal that this carcinoma shows low-grade malignancy with localization usually in the minor salivary glands. Most cases occur in women. The pathohistology is characterized by solid or trabecular formations of polygonal clear cells which are surrounded by a broad hyalinized desmoplastic connective tissue stroma. The clear cells are mucin negative and express cytokeratin and EMA, in some cases also CEA, but not S-100 protein, actin or other markers of myoepithelial cells. Ultrastructural findings are undifferentiated duct cells with only few organelles and inclusion of glycogen granules. The differential diagnosis includes other clear cell tumours, especially epithelial-myoepithelial carcinoma and the clear cell variants of myoepithelial carcinoma and acinic cell or mucoepidermoid carcinoma.

Contributions of insect systems to the chromosome theory of inheritance and to our understanding of chromosome behavior on the spindle

Insect chromosomes were among the first chromosomes to be investigated. Since then, insects have had an important place in cytogenetics. Insect studies were instrumental in proving that genes are on chromosomes and that spindle fibers exist in living cells and are not fixation artifacts. Chromosome micromanipulation studies of insect spermatocytes have provided insight into the physical properties of spindle fibers, the initial orientation (attachment) of a bivalent to the spindle, the reorientation of mal-oriented bivalents, and the cause of orientation stability. Insect spermatocytes have been important material for observing the structure of the meiotic spindle, and for determining the location and force of the chromosome motor during anaphase. In this review, I highlight important contributions of insect systems to the Chromosome Theory of Inheritance and to our understanding of chromosome behavior on the spindle.

Central nervous system neoplasms: indications for electron microscopy.

Diagnostic dilemmas of biopsy specimens in the central nervous system (CNS) tumors are often the result of multiple factors, including fixation artifact, biopsy size, lack of immunohistochemical techniques to distinguish cell types, and unawareness of rare entities. Correct diagnosis and confirmation of diagnosis of primary CNS neoplasms is imperative and may require electron microscopic examination. In some instances, use of electron microscopy may be the only approach for accurate recognition of an entity. Although diagnostic electron microscopy is expensive and cost cutting is encouraged in today's practice of medicine, cost must be weighed against the consequences of even 1 patient developing CNS treatment-related necrosis or a radiation-induced neoplasm secondary to misdiagnosis of a benign entity. This study reviews the ultrastructural differences of three groups of diagnostically difficult CNS lesions: clear cell neoplasms (ependymoma, oligodendroglioma, central neurocytoma), rare entities containing astrocytes invested by a basal lamina (pleomorphic xanthoastrocytoma, the desmoplastic neuroepithelial tumors of infancy), and benign entities characterized by transitional cell forms (subependymoma, subependymal giant cell astrocytoma).

Fast freeze-fixation/freeze-substitution reveals the secretory membranes of the gastric parietal cell as a network of helically coiled tubule A new model for parietal cell transformation

The parietal cell of the gastric mucosa undergoes rapid morphological transformation when it is stimulated to produce hydrochloric acid. In chemically fixed cells, this process is seen as a reduction in number of cytoplasmic 'tubulovesicles' as the apical surface of the cell progressively invaginates to increase the secretory surface area. It is widely believed that the tubulovesicles represent stored secretory membrane in the cytoplasm of the unstimulated cell, which is incorporated into the apical membrane upon stimulation, because they share H+,K+-ATPase activity with the apical membrane. However, fusion of tubulovesicles with the apical membrane concomitant with parietal cell activation has never been convincingly demonstrated. We have used fast freeze-fixation and freeze-substitution to study stages of morphological transformation in these cells. Tubulovesicles were not seen in the cytoplasm of any of our cryoprepared cells. Instead, the cytoplasm of the unstimulated cell contained numerous and densely packed helical coils of tubule, each having an axial core of cytoplasm. The helical coils were linked together by connecting tubules, lengths of relatively straight tubule. Lengths of straight connecting tubule also extended from coils lying adjacent to the apical and canalicular surfaces and ended at the apical and canaliculus membranes. Immunogold labelling with alpha- and beta-subunit-specific antibodies showed that the gastric H+,K+-ATPase was localized to the membranes of this tubular system, which therefore represented the configuration of the secretory membrane in the cytoplasm of the unstimulated parietal cell. Stimulation of the cells with histamine and isobutylmethylxanthine lead to modification of the tubular membrane system, correlated with progressive invagination of the apical membrane. The volume of the tubule lumen increased and, as this occurred, the tight spiral twist of the helical coils was lost, indicating that tubule distension was accounted for by partial unwinding. This exposed the cores of cytoplasm in the axes of the coils as rod-shaped elements of a three-dimensional reticulum, resembling a series of microvilli in random thin sections. Conversely, treatment with the H2 antagonist cimetidine caused severe contraction of the tubular membrane system and intracellular canaliculi. Our results indicate that tubulovesicles are an artifact of chemical fixation; consequently, they cannot have a role in parietal cell transformation. From our findings we propose an alternative model for morphological transformation in the parietal cell. This model predicts cytoskeleton-mediated control over expansion and contraction of the tubular membrane network revealed by cryopreparation. The model is compatible with the localization of cytoskeletal components in these cells.

Is Asthma a Vascular Disorder?

Is Asthma a Vascular Disorder?