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PubMed1980

Stain for skeletal muscle adenylate deaminase. An effective tetrazolium stain for frozen biopsy specimens.

Procedures for staining frozen sections of human muscle biopsy specimens for adenylate deaminase are outlined. The preeminent role of this procedure is as a survey stain for myoadenylate deaminase deficiency, a new and common muscle enzyme deficiency, an example of which is illustrated. Discrimination of type I and II muscle fibers with this stain is based on color differences rather than density differences. Color photomicrographs illustrate the variations in staining that may be encountered, and experimental controls to verify the validity of the stain are summarized.

AMP Deaminase↗

PubMed2002

Detection of submicrogram quantities of glycosaminoglycans on agarose gels by sequential staining with toluidine blue and Stains-All.

A sensitive method has been developed for the visualization of nonradiolabelled glycosaminoglycans resolved by agarose gel electrophoresis using staining with toluidine blue followed by Stains-All procedure. This method, which can detect as little as 10 ng of a single species, can be used to stain a few micrograms of a complex polysaccharide mixture. The combination of agarose gel electrophoresis and sequential toluidine blue/Stains-All staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate) and nonsulfated polyanions (i.e., hyaluronate, defructosylated capsular polysaccharide K4) as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes and the identification and quantification of the contaminations of other polysaccharides within glycosaminoglycan preparations with great sensitivity (about 0.1%). Furthermore, this method can be used to stain low-molecular-mass fractions and oligosaccharides derived from the natural polyanions, such as heparin. This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited.

Animals↗

PubMed1989

Proteins of muscle subcellular fractions in Duchenne progressive muscular dystrophy stained with "stains-all" cationic carbocyanine dye and with Coomassie Blue.

The protein compositions of subcellular fractions of muscle obtained from 17 Duchenne dystrophy patients, 15 disease controls (10 different primary myopathies, 5 spinal muscular atrophy patients), and 10 normals were examined by polyacrylamide gel electrophoresis. The gels were stained with Coomassie Brilliant Blue and with "Stains-all," which stains calcium-binding proteins, sialic acid-rich glycoproteins, and phosphoproteins. In muscle membrane fractions of Duchenne dystrophy patients there was a marked reduction in the concentrations of calsequestrin and a 39 kDa protein that stained blue with "Stains-all." There were changes in the proteins of all subcellular fractions of Duchenne's patients; some of these changes appear to be specific for Duchenne dystrophy (DD). There was no apparent correlation between the protein changes observed on acrylamide gels and the age of the patients, the duration of the disease, the degree of disability, or activity of creatine kinase. A decreased level of calsequestrin in DD sarcoplasmic reticulum may contribute to an increased level of free calcium seen in muscle from these patients.

Calcium-Binding Proteins↗

PubMed2003

[Intraoperative histological sections in obstetric brachial plexus lesions--comparison of macroscopic appearance, HE staining and toluidine blue staining].

In brachial plexus lesions and their revision, evaluation of nerve stumps is very important for the reconstructive strategy. We intraoperatively perform cryostat sections, Haematoxilin-Eosin (HE) stained, and compare the clinical appearance of the nerves to the microscopic results. Toluidine blue staining is later used to validate the structural details. Intraneural fibrosis can be traced safely with both staining methods, in root avulsions a histology is helpful, too. For more proximal, intraforaminal lesions semithin section stained with toluidine-blue are less informative than are HE-stained cryostat sections. In these lesions the clinical control by electrical stimulation and evoked potentials is superior.

Adult↗

PubMed2005

Immunohistochemical stains for p63 and alpha-methylacyl-CoA racemase, versus a cocktail comprising both, in the diagnosis of prostatic carcinoma: a comparison of the immunohistochemical staining of 430 foci in radical prostatectomy and needle biopsy tissues.

The diagnosis of prostatic carcinoma and especially minimal prostatic carcinoma can sometimes be challenging on needle core biopsy and occasionally immunohistochemistry is an aid in the diagnosis. Immunostains, such as those directed against the basal cell marker p63 and, more recently, employing antibodies reactive with alpha-methylacyl-CoA racemase (AMACR), can be useful in this situation. The aim of this investigation was to assess the diagnostic utility of a p63/AMACR antibody cocktail and compare the staining pattern it produces with that using the individual antibodies alone. A retrospective review of 31 consecutive radical prostatectomy specimens and 150 prostate needle biopsy samples was performed to select histologic sections showing foci of prostatic carcinoma and/or minimal prostatic carcinoma, high-grade prostatic intraepithelial neoplasia (HGPIN), as well as common benign mimickers of prostatic carcinoma, to include atrophy and basal cell hyperplasia, especially with prominent nucleoli. Serial histologic sections from the corresponding paraffin blocks were stained with hematoxylin and eosin and by immunostains for p63, AMACR, and a prediluted antibody cocktail comprising both. The diagnostic utility of the cocktail was assessed, and the staining characteristics it produced were compared with those using the individual immunostains. In 430 foci, the cocktail produced a p63 staining profile identical to that using the single p63 antibody. Distinction of the nuclear p63 signal from the cytoplasmic AMACR localization was readily accomplished. There was an excellent agreement (kappa=0.91; P<0.0001) between the AMACR staining profile using the cocktail and the single AMACR antibody alone. The cocktail was very useful in highlighting prostatic carcinoma associated with HGPIN, flat and cribriform HGPIN, and distorted foci of minimal prostatic carcinoma. These data indicate that use of a p63/AMACR cocktail is essentially equivalent to use of each antibody separately for immunohistochemical confirmation of a diagnosis of prostatic carcinoma in needle biopsy. This cocktail would be of diagnostic utility when only limited tissue is available for immunohistochemical evaluation of small, diagnostically difficult foci in prostate needle biopsy tissue.

Adenocarcinoma↗

PubMed1999

Rapid staining techniques in cytopathology: a review and comparison of modified protocols for hematoxylin and eosin, Papanicolaou and Romanowsky stains.

The objective of this study was to review and compare rapid protocols for fixation and staining of cytologic smears. We used fresh surgical specimens from dogs and horses to evaluate and modify, if necessary, previously described rapid staining protocols. Slides were wet-fixed, rehydrated or air-dried. Rapid Papanicolaou, hematoxylin and eosin (H&E), and Romanowsky stains were applied, including modification of Diff-Quick stain. The modified rapid staining protocols were simple to use and gave results within 5 minutes that were comparable to those obtained with traditional methods. Advantages of rehydrated vs wet-fixed smears included consistent preparations, a clean background, and equally good or superior nuclear detail.

Journal Article↗

PubMed1998

Comparison between virus isolation method, Papanicolaou stain, immunoperoxidase stain and polymerase chain reaction in the diagnosis of genital herpes.

Papanicolaou (Pap) stain, immunoperoxidase (IP) stain and polymerase chain reaction (PCR) were evaluated against the virus isolation method for their sensitivity and specificity in the diagnosis of herpes simplex virus (HSV) infection in 96 women who were suspected of genital herpes. The result showed that the sensitivity of PCR, IP and Pap stain was 100, 92.0 and 62.7%, respectively, while the specificity was 76.2, 66.7 and 81.0%, respectively. PCR was even more sensitive than the virus isolation technique. As Pap stain is the technique routinely performed for diagnosing genital herpes in most of the hospitals in Thailand, its low sensitivity should be taken into consideration. Based on the investigation by all four techniques together, HSV infection was diagnosed in 91.6% of the cases suspected of genital herpes which reflected higher precision of the clinical diagnosis over Pap stain.

Adolescent↗

PubMed1984

Selective staining of the same set of nucleolar phosphoproteins by silver and Giemsa. A combined biochemical and cytochemical study on staining of NORs.

Gel electrophoresis of nucleolar isolates from Zajdela ascites hepatoma cells followed by various staining procedures revealed a common set of bands that stained selectively with silver and Giemsa. The gel bands, corresponding to molecular weights of 104, 78, 37, and 29 kilodaltons (kd), appeared to contain phosphoproteins that were at least partly associated with oligo-deoxyribonucleotides. Enzyme digestion studies showed that the Giemsastainability was due to the phosphorylated state of the proteins. The positive selective silver-staining reaction in gels could be most likely attributed to the high content of carboxyl groups present in these phosphoproteins. The significance of these findings in relation to cytological results produced by selective silver staining of nucleolus organizing regions (NORs) and by Giemsa N-banding is discussed.

Animals↗

PubMed1985

On the nature of Romanowsky-Giemsa staining and the Romanowsky-Giemsa effect. I. Model experiments on the specificity of azure B-eosin Y stain as compared with other thiazine dye-eosin Y combinations.

After incorporation into a polyacrylamide matrix, the biopolymers DNA, RNA, heparin, hyaluronic acid, collagen and the synthetic polymers poly(U) and poly(A, U) were stained with the pure thiazine dyes, Methylene Blue, the Azures and Thionin alone and combined with Eosin Y. Satisfactory spectrophotometric agreement was obtained between the staining reactions of the biopolymers in the artificial matrix and those in their natural surroundings. This was especially true with respect to the specificity of the Azure B-Eosin Y dye-pair, which is based on the generation, on suitable substrates, of a purple colour, the Romanowsky-Giemsa effect (RGE), with an absorbance maximum near 550 nm. In the model experiments, DNA, heparin, hyaluronic acid and collagen were found to be RGE-positive and poly(U), poly(A, U) and RNA RGE-negative. A theory of RGE is proposed which complies with the new and earlier observations: after saturation of available anionic binding sites and aggregate formation by Azure B, electron donor acceptor complexes are formed between Eosin Y and Azure B via hydrogen-bridge formation of the aminosubstituent proton of Azure B and between Eosin Y and the biopolymer surface. Charge-transfer complex formation may also account for the qualitative identity of Azure B-Eosin Y and Azure A-Eosin Y spectra of substrates, which are coloured purple. Quantitatively, Azure A-Eosin Y is less efficient in giving RGE. The generation of RGE is time-dependent. Equilibrium staining is attained after about 120 h. The implications of the results for the biological application of Romanowsky-Giemsa staining are discussed briefly.

Azure Stains↗

PubMed1982

Comparative colorimetry in cytophotometric measurements of azure B-eosin Y-stained and Giemsa-stained blood cell smears.

The advantages of the Commission Internationale de l'Eclairage (CIE) color measurements and the use of the standardizable purified azure B-eosin Y stain, as compared to the commonly used commercial Giemsa stain, were studied. Color measurement and analysis according to the CIE standards allows the comparison of color measurements from different slides stained by either the same or different procedures. Moreover, measurements from different cytophotometric systems can be compared. The results can also be directly correlated with human visual subjective color estimates and thus used to quantify the human observations. The color information in the air-dried cell specimens, which differs from the color characteristics of the components of the staining solution, was measured and analyzed.

Azure Stains↗

PubMed1997

Utility of Butvar support film and methylamine tungstate stain in three-dimensional electron microscopy: agreement between stain and frozen-hydrated reconstructions.

A random conical tilt reconstruction of negatively stained Saccharomyces cerevisiae fatty acid synthase was used as a model to compute a three-dimensional reconstruction from untilted stain specimens of the molecules in multiple orientations using a three-dimensional projection alignment method. The resulting structure (24 A resolution) has a more uniform resolution than the initial structure and the handedness revealed in the random conical tilt method is preserved. In a similar approach, this model was used to compute a 21-A-resolution frozen-hydrated structure from untilted specimens of the molecules in multiple orientations. Even though the reconstructions are in close agreement, the stain structure appears to enhance the protein density associated with less robust features. These procedures significantly reduce the time and effort required to obtain a three-dimensional reconstruction from frozen-hydrated data with a resolution that is comparable to the best obtained by more laborious methods. The agreement between the stain and frozen-hydrated reconstructions affords convincing evidence concerning the validity of the structure and the information afforded by the two reconstructions significantly enhances the structural analysis of the molecule.

Coloring Agents↗

PubMed1993

A new polychrome stain and simultaneous methods of histological, histochemical and immunohistochemical stainings performed on semithin sections of Bioacryl-embedded human tissues.

We describe a new polychrome stain and simultaneous methods of histological, histochemical and immunocytochemical staining performed on sections from human tissues embedded in the new hydrophilic resin Bioacryl. The polychrome stain involves the sequential use of Harris' Haematoxylin, silver methenamine, Light Green and Eosin or Safranin dyes and provides a highly specific visualization of the overall cytological tissue architecture. When histochemical, immunocytochemical, and polychrome stains are performed together on the same section, crisp images are obtained, yielding simultaneous data of histochemical and immunological reactivities with clear tissue architecture.

Acrylic Resins↗

PubMed1975

Staining banded human chromosomes with Romanovsky dyes: some practical consequences of the nature of the stain.

Chromatographic analyses of commercial samples of Romanovsky stains were followed by various chemical and staining studies. Strong banding of chromosomes was found to be favoured by the presence in the stain of high concentrations of methylene violet Bernthsen and its immediate homologues. Chromatographic analysis showed that these dyes are present in dry Leishman and Giemsa powders only in small proportion. However, additional active dye may be generated during the process of making up staining solutions from the dry powders, if the methanol used is not contaminated with formic acid and if the period of heating is not too brief.

Chromatography↗

PubMed1985

Simultaneous demonstration of multiple antigens by indirect immunofluorescence or immunogold staining. Novel light and electron microscopical double and triple staining method employing primary antibodies from the same species.

Available techniques for light and electron microscopical double immunocytochemical staining are all associated with certain problems. We have developed a novel multiple staining procedure, which allows use of antibodies of differing specificities, raised in the same species (e.g. rabbit). Its essential features include 1) saturation of antigenic epitopes on the first layer primary antiserum by second (fluorophor- or gold-) labelled anti-IgG antibodies and 2) denaturation of free anti-IgG binding sites by formaldehyde vapour treatment. Various combinations of gastrin, somatostatin, glucagon, ACTH, growth hormone and enkephalin/endorphin antibodies have been tested at the light and electron microscopical level and have been found to give highly reproducible double- and triple-staining results. The technique has also been evaluated by use of cytochemical paper models. The method is simple and very useful for multiple staining of a wide variety of antigens.

Animals↗

PubMed1985

[Hydrophobic acridine dyes for fluorescent staining of mitochondria in living cells. 3. Specific accumulation of the fluorescent dye NAO on the mitochondrial membranes in HeLa cells by hydrophobic interaction. Depression of respiratory activity, changes in the ultrastructure of mitochondria due to NAO. Increase of fluorescence in vital stained mitochondria in situ by irradiation].

The hydrophobic fluorescence dye 10-n-nonyl-acridinium-orange-chloride, NAO, stains specifically the mitochondria of living HeLa-cells. A dye concentration of 1 X 10(-8) M is sufficient for vital staining and at 5 X 10(-7) M an incubation time less than 1 min is enough to generate the bright green fluorescence of the mitochondria. The retention of NAO by the mitochondria is longer than 7 days. The dye accumulation is not affected by the ionophores valinomycin, nigericin, gramicidin, the uncoupling agents DNP, CCCP or by ouabain. In contrast to Rh 123 the trans-membrane potential is not the driving force of the NAO accumulation. We assume that NAO is bound to the hydrophobic lipids and proteins in the mitochondrial membranes by hydrophobic interaction. With valinomycin, 500 ng/ml, 10 min, the mitochondria in HeLa-cells swell. Now it is possible to observe some details in the enlarged mitochondria by light microscopy. After vital staining with NAO, 5 X 10(-7) M, 10 min, the periphery of the swollen mitochondria shows an intense green fluorescence, the inner part is dark. Obviously the dye is bound to the membranes. By electron microscopy it can be shown that the valinomycin treated and NAO stained mitochondria have outer and inner membranes and cristae. They differ from untreated mitochondria mainly in the size. After incubation of the HeLa-cells with relatively high NAO concentrations, 5 X 10(-6) M, 10 min, the mitochondria show a weak orange fluorescence. It is generated by the dimers D of NAO. Therefore the dye concentration in the mitochondrial membranes is locally very high and causes dye dimerisation. The weak orange fluorescence is instable and disappears within a few seconds. Instead we observe a green fluorescence with growing intensity that is generated by the monomers M of NAO. The intensity has its maximum value after a few seconds. Using low NAO concentrations for incubation, 1 X 10(-7) M, 10 min, we observe only the green fluorescence with increasing intensity. In this case the orange fluorescence is too weak for observation (concentration quenching). It can be shown by experiments and quantum mechanics that the orange fluorescence is assigned to an optical forbidden, the green fluorescence to an allowed electronic transition of D or M respectively. Our results indicate a dissoziation of D in 2 M by irradiation of the mitochondria under the fluorescence microscope.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminoacridines↗

PubMed1995

Relationship between staining by digital subtraction angiography and vascularity in breast cancer: analysis of the time-density curve and the results of staining for factor VIII-related antigen/von Willebrand factor.

We performed intravenous digital subtraction angiography (IV-DSA) in 31 patients with primary invasive breast cancer, and calculated the maximum density of tumor staining from the time-density curve obtained. Specimens resected from the same patients were stained for factor VIII-related antigen/von Willebrand factor (vWF), and the amount of microvessels was calculated by image processing. A significant correlation (correlation coefficient: 0.85) was found between the maximum density of tumor staining and the vascularity determined by staining for vWF, indicating that the maximum density indirectly reflects the vascularity in the tumor.

Angiography, Digital Subtraction↗

PubMed1984

Negative staining characteristics of arrays of mitochondrial pore protein: use of correspondence analysis to classify different staining patterns.

Fourier-filtered images of negatively stained arrays of mitochondrial pore protein have been classified by correspondence analysis of their diffraction patterns. The most significant component of interpattern variation is an isotropic shift in reflection intensities between high- and low-order reflections. This corresponds in the images to the presence or absence of high resolution detail (stain minima). Experimentally the loss of detail in images correlates with the use of highly dissociated heavy metal salts as negative stains. It is proposed that such stains yield poorer negative-contrast images due to electrostatic binding of heavy-metal complex ions to fixed charge groups on the protein. Results with surface-modified protein arrays are consistent with this hypothesis.

Crystallography↗

PubMed1988

The mechanism accounted for the silver staining "fast techniques" development and its histochemical meaning. II. The binding between silver and ferric ion accounted for the silver staining development.

Results obtained on filter paper strips models show that some protein and fatty acids become argentophil as an effect of a previously binding ferric ion although, in this instance, the silver staining can only be accomplished by using a silver diamine solution, since a silver nitrate solution is not effective. However, if the filter paper model is previously treated by a "multidentate ligand" before the silver nitrate solution treatment becomes able in doing the silver staining. This result shows that the binding between Fe3+ and Ag+ can be done if a suitable negatively charged "multidentate ligand" has been connected between them. The following "multidentate ligands" were tested: thiocarbohydrazide, carbohydrazide, and hexamethylenetetramine: ammonia was also tested, as an attempt to disclose if it is able to act as a "multidentate ligand". The semicarbazide effect was analysed and compared with the "ligands". It was found, either on filter paper strip models or on histological sections, that every "multidentate ligand", as well as ammonia, are effective in producing silver staining whereas semicarbazide is devoid of effect. The "ligands" effectiveness depends upon the solution pH, in the same manner that is does occur when the silver diamine "fast technique" is used on tissue sections. Histophotometric measures, taken on tissue sections, show that the silver staining afforded by the silver diamine "fast technique" is similar to that displayed by the "multidentate ligand" plus a silver nitrate solution treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Histological Techniques↗