Simple Staining Method Research Articles

In Situ Pt Staining Method for Simple, Stable, and Sensitive Pressure-Based Bioassays.

Pressure-based bioassays (PASS) integrate a molecular recognition process with a catalyzed gas generation reaction, enabling sensitive and portable quantitation of biomarkers in clinical samples. Using platinum nanoparticles (PtNPs) as a catalyst has significantly improved the sensitivity of PASS compared with protein enzyme-based detection. However, PtNPs are easily deactivated during storage or after being decorated with antibodies. Moreover, nonspecific adsorption of PtNPs on substrates has been a problem, resulting in significant backgrounds. To solve these problems of PtNP-based detection, we report a robust, simple, stable, and sensitive Pt staining method for PASS. Detection antibody-decorated gold nanoparticles (AuNPs) are used to perform enzyme-linked immunosorbent assay, followed by Pt staining to stain AuNPs with Ag and Pt bimetallic shells (Au@AgPtNPs), which endow AuNPs with catalytic activity. The concentration of targets can be quantitatively determined by measuring the pressure due to O2 gas (g) formed by the decomposition of H2O2 catalyzed by Au@AgPtNPs. C-reactive protein and avian influenza hemagglutinin 5 neuraminidase 1 can be quantitatively detected with detection limits of 0.015 and 0.065 ng/mL, respectively. The simple, stable, and sensitive properties of the Pt staining-based method will largely broaden the applications of PASS in clinical diagnosis and biomedicine.

A Single-Step Simultaneous Protein Staining Procedure for Polyacrylamide Gels and Nitrocellulose Membranes byAlta During Western Blot Analysis.

A simple method for staining of proteins simultaneously on sodium dodecyl sulfate (SDS)-polyacrylamide gels and nitrocellulose membranes by Alta during Western blot analysis is described. A 5% solution of Alta, a commercially available cosmetic preparation, is added in the upper tank buffer during electrophoresis. On completion of electrophoresis, the gel is washed in distilled water, viewed on a white light plate and a transilluminator to photograph the protein profiles. The gel is processed for Western blot transfer of proteins onto a nitrocellulose membrane, and upon completion, the protein profiles on the membrane are viewed and photographed as stated above. The membrane can then be processed for immunostaining as per the standard procedure. Thus, the staining procedure using Alta is simple, rapid (without any need of destaining) and cost-effective.

A simple method for wrist ganglion staining with diluted surgical marking pen ink in arthroscopic resection and avoiding dye leakage-related subcutaneous discoloration

Arthroscopic resection of the wrist ganglion is commonly performed nowadays, and has a recurrence rate comparable to open excision. It is preferred by some surgeons due to the smaller and more cosmetic operative scar. When using the arthroscopic procedure, identification of the stalk of the ganglion can facilitate accurate resection. In order to achieve this, staining and enhancement of the ganglion through the use of surgical dye injection has been proposed by some surgeons. However, sterile surgical dye is sometimes not easily available, and leakage-related subcutaneous discoloration is sometimes a problem. We propose an easy method by using diluted surgical marking pen ink for wrist ganglion staining. Also, any subcutaneous leakage of the diluted dye, if it occurs, can be easily cleaned up during the arthroscopic ganglion resection.

Application of saponin on differential staining examination in animal blastocysts

Although there are several ways such as karyotyping to evaluate the quality and normality of embryos, the counting of total cell in blastocyst after the differential staining has been used as a simple indicator for quality of culture systems and normality of embryo itself. This differential staining method was regarded as a basic technique of early developmental biology of mammals, and it helps the scientific community to understand the signals regulatingmorphological events of early developmental process. The present study was undertaken to develop a simple and fast differential staining method for inner cell mass (ICM) and trophectoderm (TE) cells of mammalian blastocysts using saponin as a permeabilizing agent without using species-specific antibodies and complements. The prestained blastocyst with SYTO-13 (green) was exposed to saponin solution for propidium iodide (PI) permeation into TE cells and examined for the differential staining patterns. Three dimensional confocal microscopy was used to demonstrate the process of successful staining and showed the high impact on saponin treatment. Although the fluorescent images of blastocysts showed that one or two cell of TE stained to yellowish green, ICM was protected from saponin/PI mixture with the short exposure time of SYTO-13 pre-stained blastocysts. The total stainingprocedure did not exceed 30 min before examination under epi-fluorescence or confocal microscope. These results clearly demonstrate that saponin could be used as substituent molecule instead of species-specific antibodies and complements in differential staining examination for the first differentiation of mammalian embryos.

The CGL1 (HeLa × Normal Skin Fibroblast) Human Hybrid Cell Line: A History of Ionizing Radiation Induced Effects on Neoplastic Transformation and Novel Future Directions in SNOLAB.

Cellular transformation assays have been utilized for many years as powerful in vitro methods for examining neoplastic transformation potential/frequency and mechanisms of carcinogenesis for both chemical and radiological carcinogens. These mouse and human cell based assays are labor intensive but do provide quantitative information on the numbers of neoplastically transformed foci produced after carcinogenic exposure and potential molecular mechanisms involved. Several mouse and human cell systems have been generated to undertake these studies, and they vary in experimental length and endpoint assessment. The CGL1 human cell hybrid neoplastic model is a non-tumorigenic pre-neoplastic cell that was derived from the fusion of HeLa cervical cancer cells and a normal human skin fibroblast. It has been utilized for the several decades to study the carcinogenic/neoplastic transformation potential of a variety of ionizing radiation doses, dose rates and radiation types, including UV, X ray, gamma ray, neutrons, protons and alpha particles. It is unique in that the CGL1 assay has a relatively short assay time of 18-21 days, and rather than relying on morphological endpoints to detect neoplastic transformation utilizes a simple staining method that detects the tumorigenic marker alkaline phosphatase on the neoplastically transformed cells cell surface. In addition to being of human origin, the CGL1 assay is able to detect and quantify the carcinogenic potential of very low doses of ionizing radiation (in the mGy range), and utilizes a neoplastic endpoint (re-expression of alkaline phosphatase) that can be detected on both viable and paraformaldehyde fixed cells. In this article, we review the history of the CGL1 neoplastic transformation model system from its initial development through the wide variety of studies examining the effects of all types of ionizing radiation on neoplastic transformation. In addition, we discuss the potential of the CGL1 model system to investigate the effects of near zero background radiation levels available within the radiation biology lab we have established in SNOLAB.

Histochemical Staining of β-Glucuronidase and Its Spatial Quantification.

Microscope images of plant specimens showing expression of GUS markers, besides being very beautiful, provide useful information regarding various biological processes. However, the information extracted from these images is often purely qualitative, and in many publications is not subjected to quantification. Here, we describe a very simple quantification method for GUS histochemical staining that enables detection of subtle differences in gene expression at cellular, tissue, or organ level. The quantification method described is based on the freely available image analysis software ImageJ that is widely used by the scientific community. We exemplify the method by quantifying small and precise changes (at the cellular level) as well as broad changes (at the organ level) in the expression of two previously published reporter lines, such as the pPILS2::GUS and pPILS5::GUS. The method presented here represents an easy tool for converting visual information from GUS histochemical staining images into quantifiable data and is of general importance for plant biologists performing GUS activity-based evaluation of reporter genes.

50 ESTIMATION OF CHROMATIN ABNORMALITY OF OGYE ROOSTER SEMEN WITH Diff-Quik STAINING

The abnormality of Ogye rooster sperm chromatin could be detected by simple sperm staining. In this abstract, a Diff-Quick staining kit was tested for assessment of chicken sperm quality. Using a standard bright-field microscope, Diff-Quik stains can be reproducibly, easily, and routinely monitored with simple staining. The presence of abnormal chromatin staining of rooster sperm was determined by darker stain in head. In the fresh semen, the viabilities of 3 tested Ogye spermatozoa were 93.53, 82.42, and 90.63%, and normal chromatin rates were 87.96, 74.25, and 85.10%, respectively. However, after cryopreservation, the rates of viability of thawed semen were reduced to 69.58, 61.98, and 72.20%, and normal chromatin rate also reduced to 58.91, 48.49, and 63.34%. A significant correlation between live sperm and normal sperm nuclei was 0.875 in fresh semen and 0.513 in frozen semen. After incubation of sperm at 37°C for 5 min, the rates of viability, chromatin normality, and sperm head activity were shown as 90.63 ± 1.28%, 82.44 ± 8.09%, and 66.68 ± 10.29% in fresh semen. However, the rates of thawed semen were reduced to 67.92 ± 7.55%, 56.92 ± 12.15%, and 47.32 ± 5.02%, respectively. The relationship between chromatin normality and sperm head movements in fresh and thawed semen were 0.564 and 0.540, respectively. With these results, the chicken sperm normality could be assessed by the Diff-Quik staining, which could be used for chromatin status of sperm head and activated morphology of live spermatozoa, as a simple and rapid staining method.

Localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant® DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei of fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 106 signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy.

Distribution of gelatinous fibers in seedling roots of living cycads.

• The presence of gelatinous (tension) fibers (GFs) in the roots of two extant cycadales (Cycas and Zamia) in a recent publication raises interesting issues of GF distribution in seed plants. An immediate question that arises from this discovery is whether GFs occur consistently in the radicle of all extant cycad genera and therefore might have a similar role in root contraction. We present results of a survey of nursery-grown material of all 10 genera.• We sequentially sectioned seedling root material and used simple staining and histochemical methods to follow anatomical changes along the radicle of all 10 genera.• We found GFs in nine genera; Stangeria appears to be the only genus without them. In all genera, there is a wide variation in the number of GFs and also variation in the development of thickened, fleshy roots. "Tertiary expansion" is a useful term to describe late cell division and enlargement of both primary and secondary parenchyma, the latter produced by the vascular cambium. Certain other histological features can be diagnostically useful at the generic level.• The functional interpretation of GFs as being wholly responsible for apparent tissue contraction is now somewhat compromised, especially as distortion of tracheary elements by changes in dimensions of parenchyma cells can falsely suggest root contraction when it may not occur. These preliminary results point the way to a more precise investigation of study material grown in more uniform environments using advanced technological methods.

Diff-Quik 염색방법에 의한 오계 닭 정자의 염색질 이상과 운동성 추정에 관한 연구

Poutry Science Division, National Institute of Animal Science, RDA, Seonghwan 330-801, KoreaABSTRACT Ogye rooster sperm chromatin status can be detected using well established sperm assays. In this paper, a simple and fast method to monitor rooster sperm chromatin status could be employed in field for assessment of chicken sperm quality. Using standard bright field microscope, Diff-Quik stains can be reproducibly, easily and routinely monitored with simple staining. The presence of abnormal chromatin staining of rooster sperm was determined by darker stain in head. In the fresh semen, the viabilities of three tested Ogye spermatozoa were 93.53%, 82.42% and 90.63% and normal chromatin rates were 87.96%, 74.25% and 85.10% respectively. However, after freezing, the rates of viability of thawed semen were reduced to 69.58%, 61.98% and 72.20% and normal chromatin rate also reduced to 58.91%, 48.49% and 63.34%. A significant correlation between live sperm and normal sperm nuclei was 0.875 in fresh semen and 0.513 in frozen semen. After incubation of sperm at 37for 5min, the rates of viability, chromatin normality and sperm head activity were shown as 90.63±1.28%, 82.44±8.09% and 66.68±10.29% in fresh semen. However, the rates of thawed semen were reduced to 67.92±7.55%, 56.92±12.15% and 47.32 ±5.02%, respectively. The relationship between chromatin normality and sperm head movements in fresh and thawed semen were 0.564 and 0.540, respectively. With these results, the chicken sperm normality could be assessed by the Diff-Quik staining that could be used for chromatin status of sperm head and activated morphology of live spermatozoa, as a simple and rapid staining method.(Key words : chicken sperm, morphology, viability, Diff-Quik staining)

A facile method for simultaneously measuring neuronal cell viability and neurite outgrowth.

Neurite outgrowth is an important morphological phenotype of neuronal cells that correlates with their function and cell health, yet there are limited methods available for measuring this phenomenon. Current approaches to measuring neurite outgrowth are laborious and time-consuming, relying largely upon immunocytochemical staining of neuronal markers (e.g., beta-III tubulin or MAP2) followed by manual or automated microscopy for image acquisition and analysis. Here we report the development of a quick and simple dual-color fluorescent dye-based staining method that allows for the simultaneous measurement of neuronal cell health and relative neurite outgrowth from the same sample. An orangered fluorescent dye that stains cell membrane surfaces is used as an indirect reporter of changes in relative neurite outgrowth due to alterations in the number or length of membrane projections emanating from neuronal cell bodies. Cell viability is assessed simultaneously via the use of a cell-permeant dye that is converted by intracellular esterase activity from a non-fluorescent substrate to a green-fluorescent product. Using Neuroscreen-1 cells (a PC-12 subclone), primary rat cortex neurons, and human induced pluripotent stem cell (iPSC)-derived neurons, we demonstrate that this multiplex assay allows for rapid visualization and unbiased, quantitative plate reader analysis of neuronal cell health and neurite outgrowth.

A simple, rapid and low-cost staining method for gel-electrophoresis separated phosphoproteins via the fluorescent purpurin dye.

A novel fluorescence detection method for phosphoproteins in 1-D and 2-D SDS-PAGE by using purpurin is developed in this study. Phosphoproteins as low as 4-8 ng could be specifically detected by purpurin within 60 min, and the detection limit is similar to or better than that of Pro-Q Diamond staining. Only 2 steps (staining and destaining) are needed for purpurin staining without requiring excessive fixing and washing steps, and for single use, $0.8 is enough for purpurin staining. By comprehensively comparing with Pro-Q Diamond staining, it is concluded that purpurin staining is a simple, rapid and low-cost staining method for a broad application to the research of phosphoproteins.

A Staining Method for Assessing the Viability of Esteya vermicola Conidia

The viability of conidia of Esteya vermicola, a potentially important biocontrol agent against the pinewood nematode Bursaphelenchus xylophilus, is usually determined by cultivation for 18-48 h in culture medium. As an alternative to this labor-intensive method, we have developed a rapid, simple, and low-cost staining method for assessing E vermicola conidia survival rates. A mixture of neutral red and methylene blue was found to be the most optimal among several stains that also included safranin O and Janus green B. This mixture stained nonviable conidia blue, in contrast to viable conidia, which were stained red in the cytoplasm and blue in the cell wall. This method may be particularly useful for traditional research laboratories, as it provides rapid results using common, relatively inexpensive laboratory equipment.

A simple method for vital staining of elastin in arterial tissue.

Highly diluted solutions of Gentian Violet and Evans Blue were used to visualize the elastin network in viable porcine right common carotid artery (RCCA) preparations. The two simple, alternative methods of staining were applied to proximal, intermediate, and distal sections of RCCA under various experimental conditions. These included the state of the vessel wall soon after excision, under relaxed smooth muscle condition after preconditioning, and during vasoconstriction. Micrographs of arterial rings, sectors, and axial strips show that the RCCA is an artery of the elastic type at the proximal end and of the muscular type at the distal end. While in sections of freshly dissected or KCl-constricted arteries the elastic lamellae show the well-known waviness, those in sections from arteries with relaxed smooth muscle and after preconditioning appear nearly straight. It is hoped that the inexpensive staining tools will contribute to solve conflicting interpretations existing on elastin structures in the arterial wall.

A simple and rapid staining method for detection of hemozoin pigment by methylene blue stain.

A simple and rapid staining method for detection of hemozoin pigment by methylene blue stain.

New karyotypes of Atlantic tree rats, genus Phyllomys (Rodentia: Echimyidae)

Phyllomys (Echimyidae, Rodentia) is a genus of Neotropical rodents with available cytogenetic data restricted to six out of 13 species, mainly based on simple staining methods, without detailed analyses. In this work, we present new karyotypes for Phyllomys lamarum (diploid number 2n = 56, fundamental number or number of autosomal arms FN = 102) and Phyllomys sp. (2n = 74, FN = 140) from the state of Minas Gerais, southeastern Brazil. We provide the first GTG- and CBG-banding patterns, silver-staining of the nucleolar organizer regions (Ag-NORs), and fluorescence in situ hybridization (FISH) with telomeric and 45S rDNA probes of Phyllomys. In addition to examining their chromosomes and phenotypic characters, we sequenced mitochondrial DNA from the specimens analyzed to confirm their taxonomic identification. The comparison of the distinctive chromosome complements of our specimens with those of other species of Phyllomys already published allowed us to conclude that chromosome data may be very useful for the taxonomy of the genus, as no two species analyzed presented the same diploid and fundamental numbers (2n and FN).

Abstract B276: A live-cell apoptosis assay identifies novel combination approaches that selectively kill EGFR mutant NSCLC cells.

Abstract Introduction: Non-small cell lung cancer (NSCLC) patients with EGFR mutation positive (EGFR M+) tumours initially respond well to EGFR targeted monotherapy (e.g. gefitinib), but the benefit is usually limited due to the emergence of drug resistance. A major challenge in the field is to identify the most promising combination approaches that inhibit or prevent this resistance and then to prioritise these options for clinical testing in order to produce more durable patient benefits. Our aim in this study was to identify compounds that selectively and rapidly induced cell death but only when combined with gefitinib in EGFR M+ cells. Methods: We developed a high-throughput 96-well live-cell phenotypic assay (based on an acute apoptosis end point) to assess the combination potential of probe inhibitors targeting kinases (n=402), as well as compounds that are approved or in clinical development for the treatment of cancer (n=326). Screens were carried out using EGFR M+ (PC-9, HCC-827, HCC-2935, H1975) and EGFR WT (A549, H1993) NSCLC cells, and a non-tumor immortalized lung epithelial cell line (BEAS2B). Cells were treated with gefitinib (0-1 µM) and test compounds (0-10 µM) for 18 h. Hoechst 33258 (25 µM) was added and 30 min later cells with condensed nuclei and a high nuclear intensity were scored as apoptotic, and the percentage of apoptotic cells was calculated. SYTOX green DNA staining confirmed that apoptotic cells were scored positive using our simple Hoechst staining method. Results: The screening assay was robust and reproducible with Pearson correlation coefficients ranging from 0.66-0.92 when comparing inter-assay apoptosis scores. Using the compound library we identified 21 positive hits (Z-score≥10) that selectively induced apoptosis in the presence of gefitinib in EGFR M+ cells, but not in EGFR WT cells. Positive hits included compounds in four major target categories; BCL inhibitors (n=3), anti-mitotic agents (n=11), HDAC inhibitors (n=4), and AKT/PI3K inhibitors (n=5). Several of these targets have been suggested in the literature using different methodologies to our own, thereby validating our overall screening approach. Importantly, we have also identified several previously unreported combinations that induced marked apoptosis within 18 h of gefitinib treatment, and which we are currently evaluating in in vivo studies. In addition, we observed that combinations of EGFR TKIs with gefitinib were antagonistic in EGFR M+ cells. Finally, in the probe inhibitor library set, we have identified 20 diverse chemical structures that are strongly positive in our combination assay, suggesting new targets for selective killing of EGFR M+ NSCLC cells. Unexpectedly, screening the chemical probe library also identified 7 compounds that selectively induced cell death in combination with gefitinib in EGFR wild-type KRAS mutant A549 cells. Conclusion: We have developed a robust and reproducible high-throughput live cell apoptosis assay to screen compound libraries in combination with gefitinib in NSCLC cells. We have identified several novel combinations that are highly selective for EGFR M+ cells in the presence of gefitinib. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B276. Citation Format: Sivan M. Bokobza, Aoife M. Devery, Anika M. Weber, Anderson J. Ryan. A live-cell apoptosis assay identifies novel combination approaches that selectively kill EGFR mutant NSCLC cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B276.

Rapid staining method to detect and identify downy mildew (Peronospora belbahrii) in basil.

• Premise of the study: Demand for fresh-market sweet basil continues to increase, but in 2009 a new pathogen emerged, threatening commercial field/greenhouse production and leading to high crop losses. This study describes a simple and effective staining method for rapid microscopic detection of basil downy mildew (Peronospora belbahrii) from leaves of basil (Ocimum basilicum).• Methods and Results: Fresh leaf sections infected with P. belbahrii were placed on a microscope slide, cleared with Visikol™, and stained with iodine solution followed by one drop of 70% sulfuric acid. Cell walls of the pathogen were stained with a distinct coloration, providing a high-contrast image between the pathogen and plant.• Conclusions: This new staining method can be used successfully to identify downy mildew in basil, which then can significantly reduce its spread if identified early, coupled with mitigation strategies. This technique can facilitate the control of the disease, without expensive and specialized equipment.

Highly Selective and Fast Diagnosis of Alzheimer's Disease Hallmark Lesions using Congo Red in Isopropyl Alcoholic Solution

A highly selective, rapid, inexpensive, simple, and immunocytochemical compatible fluorescence staining method for Alzheimer's disease hallmark lesions applicable to sections of human specimens embedded in paraffin is described. Human necropsy material was fixed in buffered formalin, sectioned at 10 μm, mounted on slides, deparaffinized, and partially hydrated (70% ethanol). After partial hydration, sections were stained for 10 min in a solution of 0.2% Congo red in 70% isopropanol. After washing in 70% isopropanol and rehydration, auto-fluorescence of sections were quenched (optional) and processed for immunocytochemistry (optional). Finally, sections were mounted in an adequate mounting medium. Amyloid deposits appear pink at light microscopy and all Alzheimer's disease hallmark lesions appear orange or red under fluorescence microscopy using blue or green exciting light, respectively. The present method can be used in combination with all pre- or post-immunocytochemical techniques.

Measuring Cr Volatility from Ferritic Stainless Steels: Novel and Conventional Methods Compared

Reactive evaporation of Cr-species from ferritic stainless steels is a technical challenge for solid oxide fuel cell (SOFC) systems and other devices operating in high-temperature (>500°C) oxidizing environments. A traditional method for quantitatively measuring Cr volatility is by using a transpiration system, in which oxidizing gases are flown by Cr-containing materials and the exhaust is condensed and subsequently analyzed using ICP-MS. While this method is well established and accurate, it also has limited sensitivity and challenges associated with sample collection. Novel methods have recently been developed to quickly and accurately evaluate and quantify low levels of Cr volatility. These include the denuder technique, which employs a water-soluble reactive collection and optical analysis; a cold quartz wool collection method with ICP-MS; a cold Si-wafer collection with ion-beam analysis; an ionic conductivity measurement of condensed exhaust solution; and, a simple staining method. Each method has advantages and disadvantages in terms of cost, operation, sensitivity and accuracy. In this poster, Cr volatility measurement techniques will be presented and compared in the context of facilitating new and ongoing research efforts.