Developed Staining Method Research Articles

Detection of neutrophil extracellular traps formation using the hexaazotated fuchsin and determination of the immunoglobulin classes A and M abzyme activity in patients with bronchial asthma and COPD

Netosis and abzyme activity are the significant components of the innate immunity. Spontaneous and induced netosis, various types of proteolytic and oxidoreductase abzyme activity in patients with COPD and BA were determined in this study. NETs were determined by both light and fluorescence microscopy using a previously developed staining method. As a result of the work, an increase of the induced netosis was found in patients with COPD, relative to patients with BA. A significant increase in the IgA peroxidase activity was found in patients with BA relative to patients with COPD. Data on hyaluronidase, catalase, elastase, dipeptidyl dipeptidase-4-similar activity were obtained. A correlation between the peroxidase and catalase activity of IgA in patients with BA was found.

Fluorescent characterization of amyloid deposits in the kidneys of mdx mice.

An amyloidosis is a group of diseases that occurs when amyloid proteins are deposited in tissues and organs. In this study, we demonstrated, at the first time, the presence of amyloid deposits in the kidneys of dystrophin-deficient mdx mice, which are widely used as an experimental model of Duchenne muscular dystrophy. We examined cases of renal amyloidosis in aged mdx mice using traditional methods for amyloid detection (Congo red and Thioflavin T), as well as a new fluorescent dye, disodium salt of 2,7-(1-amino-4-sulfo- 2-naphthylazo) fluorene (DSNAF). Using these different fluorescent dyes, we confirmed the amyloid structure of protein deposits in kidneys of aged mdx mice. Additionally, we found that fixation method has profound effects on downstream staining procedures, and demonstrated that the application of specific fixative, zinc-ethanolformaldehyde, instead of traditional neutral-buffered formalin allows reducing the background fluorescence. Also we illustrated the usefulness of novel fluorescent dye DSNAF for detection of amyloid deposits in mouse tissues. Taken together, our results indicate the association of amyloid formation with Duchenne muscular dystrophy and provides new opportunities for studying naturally occurring amyloidosis development in tissues also.

An ultrasensitive stain for negative protein detection in SDS-PAGE via 4′,5′-Dibromofluorescein

A highly sensitive method for brief and economical staining of proteins in 1-D and 2-D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by 4′,5′-Dibromofluorescein (DBF) was developed in this study. Down to 0.025–0.05ng protein could be detected within 10min (only 2 steps) by DBF stain, which is approximately 10-fold more sensitive than those of Eosin Y (EY) and SYPRO Ruby stains, and 20-fold more sensitive than that of imidazole-zinc (IZ) negative stain. In addition, the LC-MS/MS results indicated that the newly developed staining method is compatible with the downstream protein identification. Moreover, the mechanism of DBF stain was investigated by molecular docking.

Positive staining for cellulose in oral pulse granuloma

Oral pulse granuloma (OPG) is an oral inflammatory lesion characterized by the presence of hyaline rings with numerous multinucleated giant cells. The etiopathogenesis of this lesion is thus far unclear, as is the composition of the hyaline rings. Our aim was to investigate whether the hyaline rings contain cellulose. Using a newly developed staining method for cellulose, we studied 18 histologic samples diagnosed as OPG, in addition to 3 samples originally diagnosed as "normal" foreign body reactions. In our study, visualization of cellulose is based on its specific binding to the carbohydrate binding module of β-1,4-glycanase. All samples diagnosed as OPG were positive for cellulose staining localized in hyaline rings. In addition, 1 lesion (of3), first diagnosed as a foreign body reaction without the presence of hyaline rings, was positive for cellulose by horseradish peroxidase staining. We show for the first time that cellulose is present in OPG lesions, indicating that cellulose might be the initial cause of formation of these lesions.

Highly Sensitive Method for Specific, Brief, and Economical Detection of Glycoproteins in Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis by the Synthesis of a New Hydrazide Derivative

A new hydrazide derivative was synthesized and used for the first time as a specific, brief, and economical probe to selectively visualize glycoproteins in 1-D and 2-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with high sensitivity. The detection limit of the newly developed staining method is 2- and 4-fold higher than that of the widely used Pro-Q Emerald 300 and 488 stains, respectively.

A high throughput Nile red fluorescence method for rapid quantification of intracellular bacterial polyhydroxyalkanoates

A rapid quantitative measurement of accumulated polyhydroxyalkanoate (PHA) is essential for rapid monitoring of PHA production by microorganisms. In the present study, a 96-well microplate was used as a high throughput means to measure the fluorescence intensity of the Nile red stained cells containing PHA. The linear correlation obtained between intracellular PHA concentration and the fluorescence intensity represents the potential of the Nile red method employment to determine PHA concentration. The optimal ranges of excitation and emission wavelengths were determined using bacterial cells containing different types of PHAs, of different co-monomers and compositions. Interestingly, in spite of different co-monomers compositions in each PHA, all tested PHAs fluoresced maximally at excitation wavelength between 520 and 550 nm, and emission wavelength between 590 and 630 nm. The developed staining method also had successfully demonstrated a good correlation between the amount of accumulated PHA based on the fluorescence intensity measurements and that from chromatographic analysis to evaluate poly(3-hydroxybutyrate) [P(3HB)], poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)], using the same calibration curve, despite of different co-monomers that the PHA consist. Strongly supported by these experimental results, it can therefore be concluded that the developed staining method can be efficiently applied for rapid monitoring of PHA production.

Ectoine production by Alkalibacillus haloalkaliphilus—Bioprocess development using response surface methodology and model‐driven strategies

Halophile microorganisms are important ectoine producers and are applied in industrial applications. The growth and osmoregulation are distinguished based on the diversity of environmental distribution and require holistic investigations of cultivation condition and osmotic response. The barely investigated halophilic bacterium Alkalibacillus haloalkaliphilus is able to accumulate the compatible solute ectoine, while growing under environmental stress conditions. Since microbial growth and ectoine production are functions of the process key parameters such as salinity, pH value, and temperature, the optimization resulted in three significant process strategies that were estimated by response surface methodology. For each strategy, single‐cell analysis was performed using a newly developed staining method for monitoring the membrane potential of halophilic microorganisms directly in the cultivation medium. The flow cytometric method was shown to reveal the physiological state during ectoine production and has a great potential to be applied for basic research, process optimization, and quality control.

An expanding negative detection method for the visualization of DNA in polyacrylamide gels by eosin Y

A sensitive and easy technique has been developed for the negative detection of DNA following PAGE using eosin Y. After electrophoresis, gels are fixed and stained within 40 min to provide a detection limit of 0.1-0.2 ng of single DNA band, which appears as transparent and colorless under the opaque gel matrix background. The sensitivity of the new stain is fourfold better than zinc-imidazole negative and ethidium bromide stains. Furthermore, the newly developed staining method has been successfully applied to RNA visualization in polyacrylamide gels. In addition, the inclusion of inorganic salts in staining solution was also investigated, which has great effect on the staining efficiency.

Dynamics in bacterial surface properties of a natural bacterial community in the coastal North Sea during a spring phytoplankton bloom

The hydrophilic and hydrophobic properties of single cells of natural bacterioplankton communities were determined using a recently developed staining method combined with confocal laser scanning microscopy and advanced image analysis. On an average, about 50% of the bacterial cell area was covered by hydrophobic and only 16% by hydrophilic properties, while about 72% was covered by the genome. However, the size of these properties was independent of the bacterial cell size. Bacterial hydrophobicity was positively correlated with ambient NH 4 + concentrations and negatively correlated with overall bacterial abundance. The expression of hydrophilicity was more dynamic. Over the spring phytoplankton bloom, the bacterioplankton ratio phil/phob repeatedly reached highest values shortly before peaks in bacterioplankton abundance were observed, indicating a direct and fast response of bacterial surface properties, especially hydrophilicity, to changing environmental conditions. Compared to bacterial strains, recently studied with the same method, cells of marine bacterioplankton communities are much smaller and less frequently covered by hydrophobic or hydrophilic properties. While the percentage area covered by the genome is essentially the same, the percentage area covered by hydrophobic or hydrophilic properties is much smaller.

The morphology of semicrystalline segmented poly(ether ester) thermoplastic elastomers

AbstractThe supermolecular structure of melt‐crystallized segmented copoly(ether ester)s based on poly(tetramethylene terephthalate) and poly(tetramethylene oxide) is investigated by polarizing microscopy and electron microscopic techniques. Spherulites are formed on isothermal crystallization at supercoolings ΔTu < 30°C. The spherulites recrystallize to form dendritic structures on further annealing at higher temperatures. The spherulites and dendrites are builtup from lamellae which exhibit little variation concerning the thickness of their crystalline core. The internal phase boundaries in these systems are made visible with a newly developed staining method. A network of interlocked individual lamellae is formed on rapid quenching of the melt films. Row crystallization (shish‐kebabs) is observed, if the melt is sheared during the crystallization. It is concluded from these observations that the chemical structure of the segmented copolymers bears little relevance for the supermolecular structure of these materials except for the thickness of the crystalline core of the lamellae which seems to be related to the molecular length of the hard segment sequences occurring with the highest concentration in the sample.