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Olfactory neuroblastoma. Cytodiagnostic features in a case with ultrastructural and immunohistochemical correlation.

In a case of olfactory neuroblastoma, originally misdiagnosed as an undifferentiated carcinoma, cytologic examination of material scraped from the superior nasal vault revealed tumor cells suggestive of neuroblastoma. The most significant cytodiagnostic feature was the presence of a fibrillary cytoplasm with ill-defined borders. Also noteworthy were the smudged hyperchromatic nuclei and structures resembling rosettes or pseudorosettes. The diagnosis was confirmed by electron microscopy, which revealed the presence of dense-core neurosecretory granules, clear vesicles, neurotubules and neurofilaments, and by immunohistochemistry, which showed positive staining for neuron-specific enolase but negative staining for keratin and glial fibrillary acidic protein. Since olfactory neuroblastoma has a relatively good prognosis and aggressive surgical resection may be curative, it is important that this tumor be distinguished from other small cell malignancies arising in the nasal cavity. The present case shows that the diagnosis can be made by the cytologic examination of scrapings from the tumor.

Aged↗

Assessment of immunocytochemical and histochemical stainings in the distinction between reactive mesothelial cells and adenocarcinoma cells in body effusions.

BACKGROUND: The accumulation of fluid in body cavities is a common event in both neoplastic and non-neoplastic diseases. However, the distinction between cells of reactive process and those of malignancy in cytology is not always possible. It is especially difficult when reactive mesothelial cells and adenocarcinoma cells are encountered. The aim of the present study was to find out the most accurate or reliable immunocytochemical and histochemical stains to distinguish reactive mesothelial cells from adenocarcinoma cells, and to serve as a standard method in the future when dealing with equivocal cases. METHODS: Ninety-nine cases of malignant epithelial effusion were collected from 755 cases of effusion obtained from 3 large body cavities in the past one-year period. Among them, 71 cases were histologically as adenocarcinoma and 13 cases as non-adenocarcinoma. The other 15 cases were carcinoma proved by image as well as clinical symptoms and signs. These 99 cases, plus 10 cases of non-malignant effusion, underwent immunocytochemical and histochemical stainings. Five common commercial antibodies used in this immunocytochemical study were epithelial membrane antigen (EMA), carcinoembryonal antigen (CEA), cytokeratin, vimentin and Leu-M1. The histochemical study included periodic acid-Schiff diastase (D-PAS) and mucicarmine stains. RESULTS: The immunocytochemical study showed that EMA had a high frequency of positive staining with malignant epithelial cells and a negative staining with mesothelial cells. Cytokeratin always stained with malignant epithelial cells but it also stained with mesothelial cells. Almost all the proved adenocarcinomas expressed CEA which was not expressed in the proved non-adenocarcinomas. However, CEA occasionally stained mesothelial cells. Leu-M1 showed a low frequency of staining with the proved adenocarcinoma cells, but it did not stain proved non-adenocarcinoma cells and mesothelial cells. On the contrary, vimentin stained with all mesothelial cells but occasionally with malignant epithelial cells, especially the proved adenocarcinoma cells. In the histochemical study, both mucicarmine and D-PAS showed a low sensitivity but high specificity in detecting the adenocarcinoma cells. CONCLUSIONS: No single marker is absolutely reliable to distinguish exfoliated, reactive mesothelial cells from adenocarcinoma cells in effusions. However, a panel of 3 antibodies containing EMA, CEA and vimentin, together with D-PAS and mucicarmine stains may help solve this problem.

Adenocarcinoma↗

Lectin immunohistochemistry study of nasal inverted papilloma and associated neoplasms.

BACKGROUND: We aimed to investigate the tumor biology of nasal IP and identify the biological characteristics associated with early diagnosis and malignant transformation. MATERIALS AND METHODS: Lectins immunohistochemistry were performed on inverted papilloma (IP), and associated neoplasms. IP synchronized with polyp or squamous cell carcinoma (SCC) and IP metachronized with SCC were analyzed and correlated to clinical information. RESULTS: Canavalia ensiformis (ConA) and Arachis hypogaea (PNA) with neuraminidase pretreatment (NA-PNA) showed similar staining in both the IP and SCC portions of the IP synchronized with SCC. The IP and polyp portions of an IP synchronized polyp had positive NA-PNA staining, while papilloma and polyps alone had negative staining. Strong NA-PNA staining in the IP (transformed to SCC) showed significant differences from IP. CONCLUSION: These biological characteristics define IP as a premalignant neoplasm. NA-PNA staining may be helpful for an early detection of IP. Strong NA-PNA staining in IP may predict malignant transformation.

Adult↗

In vivo presence of capsular polysaccharide in coagulase-negative staphylococci of ovine origin.

Three strains of coagulase-negative staphylococci identified as Staphilococcus warnery, Staphylococcus simulans and Staphylococcus haemolyticus were examine for evidence of capsular polysaccharide, in vitro by negative staining with India ink, and in vivo by transmission electron microscopy. In vitro, unstained materials surrounded clusters or single cocci. In vivo, capsula materials were surrounding phagocytosed bacteria cells. These capsular materials were either closely or loosely bound to bacterial cells, or projecting like "pili" from the surface of the cell wall. The evidence suggests that capsular polysaccharide is a common characteristic of coagulase-negative staphylococci causing prolonged intramammary infection of sheep.

Animals↗

Biophysical analysis of normal transthyretin: implications for fibril formation in senile systemic amyloidosis.

Transthyretin (TTR) is a plasma protein that transports thyroid hormone and retinol binding protein-vitamin A complex. Eighty-four variants of TTR have been identified and seventy-four are associated with familial amyloidotic polyneuropathy. Normal TTR is the major protein found in the fibrillar deposits in the heart at time of autopsy of individuals with senile systemic amyloidosis. The mechanism by which normally soluble TTR deposits as organ-damaging, insoluble, pathological fibrils late in life is unknown. Understanding the mechanism of fibrillogenesis of normal TTR is critical to the design of clinical treatments aimed at retardation, prevention, or reversal of fibril deposition. We have employed a biophysical approach to explore the hypothesis that an instability in a particular secondary or tertiary structure plays a role in the ability of normal TTR to form fibrils at physiological pH. Using far UV circular dichroic (CD) spectroscopy as a function of temperature we have identified simultaneous, cooperative, reversible structural changes in the beta-sheet and alpha-helical regions. The flexible short, surface-located loops undergo an irreversible conformational change at a lower temperature. Spectra before and after heating are different, particularly in the wavelength region associated with these loops, strongly suggesting that the major portion of TTR returns to its initial conformation while the loops do not. Near UV CD reveals partially reversible and irreversible changes in tertiary structure. Using calorimetry to directly measure the enthalpy associated with these changes, two peaks are observed, with further analysis suggesting conformational intermediates. Precipitates from heated samples reveal pre-fibrillar morphology by negative stain electron microscopy. These biophysical studies suggest that heat-induced conformational rearrangements enable normal TTR to assemble into pre-fibrils at physiological pH.

Amyloid Neuropathies, Familial↗

Prospective evaluation of AMACR (P504S) and basal cell markers in the assessment of routine prostate needle biopsy specimens.

Distinguishing benign prostate glands from malignant ones, based purely on morphology, on prostatic core needle biopsy specimens (PNBs) may prove difficult, particularly if the suspicious focus is small. In recent years, several immunohistochemical markers, including the basal cell cocktail (BCC), 34betaE12 and p63, and the prostate cancer (PCa) biomarker alpha-methylacyl-CoA-racemase (AMACR), have been used as adjuvants to morphology, in these diagnostically challenging cases. We prospectively address the diagnostic utility of using the BCC, in combination with the commercially available AMACR monoclonal antibody, P504S, on PNBs that required immunohistochemistry (IHC) studies to make a diagnosis. The goals of this prospective study were to assess the day-to-day practice in an academic setting, to determine how often these IHC tests were used on routine PNBs, and to establish how often a combination of the BCC and P504S were helpful in diagnosing prostate cancer. A total of 772 prospectively collected PNB cases were examined over a 7-month period. IHC staining was performed in 171 cases (22%); 123 cases were stained with the BCC in addition to the commercially available monoclonal AMACR antibody. In 86 of these 123 cases (70%), both stains contributed to the final diagnosis: PCa in 44 cases, benign in 33 cases and high-grade prostatic intraepithelial neoplasia in 9 cases. Of the remaining 37 cases (30%), 18 were called benign or PCa, based solely on appropriate staining with the BCC, with AMACR being noncontributory because the focus of interest had been cut through (12 cases), there was negative staining with AMACR (in 4 PCa cases), or there was positive staining with AMACR (in 2 benign cases showing atrophy). Nineteen of 37 cases were diagnosed as atypical small acinar proliferation. In these 19 cases either the focus had been cut through on one or both of the stains (11 cases), both AMACR and BCC failed to work (2 cases), AMACR was positive in the presence of patchy BCC staining (1 cases), AMACR was negative in the absence of BCC staining (3 cases), or despite appropriate staining the focus consisted of 1 gland and was considered too small to call carcinoma (2 cases). Additional IHC stains were performed in 171 of 772 cases; of these, 123 had sufficient material to perform both the BCC and P504S. The BCC when used in combination with AMACR rendered a diagnosis in almost 70% of cases. Using these stains in combination may be a better approach in diagnostically difficult cases as it increases the likelihood that a definitive diagnosis can be rendered while decreasing the likelihood of an equivocal diagnosis. However, a limitation of this approach is the loss of tissue in these small lesions, suggesting that combining AMACR and the BCC on a single slide would be superior to using either marker separately.

Adenocarcinoma↗

Morphological estimation of total number of influenza A type virion spikes.

When purified and stored influenza A type virions were observed by the negative staining method in electron microscopy, we found disintegrated virions associated with a population of dispersed but clustered spikes. The total spike numbers of the clusters were morphologically counted, and their peak number (about 200) was consistent with the spike number measured by the molecular biology method, described in the other report.

Influenza A virus↗

Trichomonas vaginalis: observation of coexistence of multiple viruses in the same isolate.

Trichomonas vaginalis is a flagellated, parasitic protozoan that inhabits the urogenital tract of humans. Some isolates of T. vaginalis are infected with a double-stranded RNA (dsRNA) virus, which was described in the literature as homogeneous icosahedral viral particles with an isometric symmetry and 33 nm in diameter. This study examined in detail the viral particles in T. vaginalis isolate 347 and describes a heterogeneous population of viral particles. The different dsRNA viruses were only observed after a change in the technique. The sample was prepared by the negative staining carbon-film method directly onto freshly cleft mica. The detected viruses ranged in size from 33 to 200 nm. Among the shapes observed were filamentous, cylindrical, and spherical particles. These results show that T. vaginalis may be a reservoir for several different dsRNA viruses simultaneously.

Animals↗

Special specimen preparation methods for image processing in transmission electron microscopy: a review.

One of the important developments in quantitative electron microscopy has been the application of optical and computer imaging methods to electron micrographs. In general these techniques of image analysis have been applied to electron micrographs from isolated biological structures prepared in the presence of various negative stains. To make full use of image processing techniques there are obvious advantages in preparing suitable specimens containing large areas of repeating features. However, the number of naturally occurring biological specimens exhibiting crystalline or paracrystalline features suitable for high resolution electron microscopy and subsequent image analysis is relatively small.Some recent experiments on the in vitro formation of crystalline and paracrystalline arrays from highly concentrated and purified isometric, filamentous and rod-like viruses is reviewed. The problems associated with the preparative procedures for producing two-dimensional and three-dimensional crystalline arrays are discussed together with the possibility of extending the negative staining-carbon film method for studying the gradual dissociation or assembly of viral components.

Computers↗

Poor development of outer dense fibers as a major cause of tail abnormalities in the spermatozoa of asthenoteratozoospermic men.

For a reasonable analysis of sperm motility, consideration should be given to the morphology of the flagellum. Among various tail disturbances, the present paper describes a structural shaft defect that is characterized by variations in diameter, poor outline and/or lacking endpiece. Electron microscopical examinations, especially the negative staining technique, have shown that in addition to membrane alterations there is one essential cause for the limited extension of the outer dense fibres. This disturbance may be of genetic origin or due to toxic or inflammatory influences. Further molecular biological studies are required to detect the actual cause of this defect, for which there is so far no adequate therapy.

Humans↗

Electron microscopy of the actin-myosin head complex in the presence of ATP.

The structure of the actin-myosin head complex during the ATPase cycle has been studied by electron microscopy of negatively stained acto-heavy-meromyosin. In the absence of ATP, heavy meromyosin molecules generally showed a regular, angled appearance, with both heads attached to the actin filament. In the presence of ATP, attached molecules showed a less ordered structure, often with only one head attached. We conclude that configurations other than the rigor structure occur during the actomyosin cross-bridge cycle.

Actins↗

[Expression of ras oncogene product P21 in thyroid tumors: an immunohistochemical study].

The p 21 product of ras oncogene has been detected immunohistochemically in normal, inflammatory, benign and malignant human thyroid tissues. With the monoclonal antibody SCI-oncogene I and an avidin-biotin-peroxidase complex (ABC), the expression of ras p 21 was evaluated in paraffin-embedded sections. The results showed that papillary and follicular adenocarcinomas of the thyroid had moderate to intense staining for ras p 21 in most cases. Cytoplasmic and apical surface staining were the most common patterns of immunoreactivity. Adenomas showed slight positive or negative staining in cytoplasm. Normal thyroid tissues and thyroiditis were uniformly negative. Grave's disease revealed slight to moderate staining in some cases. These findings suggest that ras oncogene is involved in carcinogenesis of thyroid carcinomas. Enhanced expression of ras p 21 may be useful in differentiation of thyroid adenocarcinomas from adenomas and may be a valuable parameter in evaluating biological behavior of tumors.

Adenocarcinoma↗

An x-ray scattering study of bromegrass mosaic virus.

X-ray scattering data and electron microscope observations are presented for bromegrass mosaic virus. Its radial density distribution is obtained from the Fourier transform of the amplitudes of the scattered x-rays. The results indicate that the virus is 260 A in diameter, it has an almost empty central cavity which is about 80 A in diameter, and the regions occupied by RNA and protein are approximately equal in average density. Electron micrographs of negatively stained preparations also give an outside diameter of 260 A and indicate that there is a central region about 90 A in diameter into which uranyl acetate can penetrate. Positively stained preparations indicate that the nucleic acid is concentrated in a shell-shaped region which is in turn surrounded by a shell of protein. In order for the RNA and protein regions to have the same average electron density the RNA must have a hydration of 1.29 gm of water per gm of RNA and the protein must have a hydration of 0.24 gm of water per gm of protein.

Bromovirus↗

POLYOMA-LIKE VIRIONS IN HUMAN DEMYELINATING BRAIN DISEASE.

Specimens of brain tissue obtained at autopsy from three patients suffering from progressive multifocal leukoencephalopathy (PML) were examined by electron microscopy. In specimens from all three cases particles similar to those of the papova virus group were present, confirming previous observations. By the negative staining method it was possible to define the morphological characteristics of the particles more precisely and it was shown that they are structurally similar to virions of the polyoma-SV40-K type. The need is emphasized for obtaining fresh unfixed diseased tissue from persons suffering from PML in order that the biological properties of the particles can be investigated.

Autopsy↗

Expression of L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in liver tumor lesions of rat models.

BACKGROUND AND OBJECTIVES: It has been said that amino acid transporters play an important role in supplying nutrition to cells and for cell proliferation. In this study, we examined whether LAT1 and 4F2hc are closely related to tumor growth. METHODS: Rat colon cancer cells (RCN-9) were injected into the spleen of 12 male rats (inbred F344/DuCrj). In each rat, liver samples including tumor lesions were immunostained with anti-LAT1 and anti-4F2hc antibodies. The staining area of LAT1 and 4F2hc tumor lesions was calculated by computer analysis. RESULTS: Sixty-eight tumor nodules were observed in 12 livers. Out of the 68 tumor nodules, 36 nodules (52.9%) indicated a positive staining of LAT1 and 32 (47.1%) had a negative staining of LAT1. However, the LAT1 expression was scarcely detected in non-tumor areas. In terms of the 4F2hc expression, there were 56 nodules (82.4%) with 4F2hc positive and 12 (17.6) with 4F2hc negative. In addition, the expression of 4F2hc in non-tumor areas was almost the same as the expression of 4F2hc in tumor lesions. The average tumor size of the group with LAT1 positive and 4F2hc positive (n = 31) was 0.845 +/- 0.232 mm(2), which was significantly larger than that of the group with LAT1 negative and 4F2hc negative group (n = 7) (0.090 +/- 0.028 mm(2)) or the group with LAT1 positive and 4F2hc negative (n = 5) (0.097 +/- 0.025 mm(2)), respectively (P = 0.0017, P = 0.007). CONCLUSION: LAT1 was related to tumor growth. We think that LAT1 can possibly enhance its ability to promote tumor growth in cooperation with 4F2hc.

Animals↗

A structural analysis of the regularly arranged porin on the outer membrane of Campylobacter jejuni based on correlation averaging.

A negatively stained electron micrograph of regularly arranged porin proteins of Campylobacter jejuni on the isolated outer membrane of bacteria was analyzed in detail by the correlation averaging method using a computer-assisted program. The results showed that the porin of C. jejuni had a trimeric structure separated by about 10.4 +/- 0.15 nm. In addition, the pores in the trimers were also separated by about 4.3 +/- 0.1 nm.

Campylobacter jejuni↗

The chicken muscle thick filament: temperature and the relaxed cross-bridge arrangement.

Although chicken myosin S1 has recently been crystallized and its structure analysed, the relaxed periodic arrangement of myosin heads on the chicken thick filament has not been determined. We report here that the cross-bridge array of chicken filaments is temperature sensitive, and the myosin heads become disordered at temperatures near 4 degrees C. At 25 degrees C, however, thick filaments from chicken pectoralis muscle can be isolated with a well ordered, near-helical, arrangement of cross-bridges as seen in negatively stained preparations. This periodicity is confirmed by optical diffraction and computed transforms of images of the filaments. These show a strong series of layer lines near the orders of a 43 nm near-helical periodicity as expected from X-ray diffraction. Both analysis of phases on the first layer line, and computer filtered images of the filaments, are consistent with a three-stranded arrangement of the myosin heads on the filament.

Actin Cytoskeleton↗

The conformation of DNA packaged in bacteriophage G.

When packaged in a bacteriophage capsid, double-stranded DNA occupies a cavity whose volume is roughly twice the volume of the DNA double helix. The data thus far have not revealed whether the compactness of packaged bacteriophage DNA is achieved by folding of the DNA, undirectional winding of the DNA, or a combination of both folding and winding. To assist in discriminating among these possibilities, the present study uses electron microscopy, together with ultraviolet light-induced DNA-DNA cross-linking, to obtain the following information about the conformation of DNA packaged in the comparatively large bacteriophage, G: 1) At the periphery of some negatively stained particles of bacteriophage G, electron microscopy reveals standards of DNA that are both parallel to each other and parallel to the polyhedral bacteriophage G capsid. However, these strands are not visible toward the center of the zone of packaged DNA. 2) Within some positively stained particles, electron microscopy reveals DNA-associated stain in relatively high concentration at corners of the polyhedral bacteriophage G capsid. 3) When cross-linked DNA is expelled from its capsid during preparation for electron microscopy, some DNA molecules consist primarily of a compacted central region, surrounded by DNA strands that appear to be unravelling at multiple positions uniformly distributed around the compacted DNA region. The above results are explained by a previously presented model in which DNA is compacted by folding to form 12 icosahedrally arranged pear-shaped rings.

Bacteriophages↗