scholarly journals An improved DAPI staining procedure for visualization of polyphosphate granules in cyanobacterial and microlagal cells

2015 ◽  
Author(s):  
Chandan Mukherjee ◽  
Chandan Mukherjee ◽  
Krishna Ray
Keyword(s):  
Staining Procedure ◽  
Dapi Staining ◽  
Polyphosphate Granules

Ca-Histochemistry in slime mold plasmodia

1978 ◽  
Vol 36 (2) ◽  
pp. 130-131
Author(s):  
Ulrich Dierkes
Keyword(s):  
Physarum Polycephalum ◽  
Carbon Coating ◽  
Freeze Drying ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Slime Mold ◽  
Staining Procedure ◽  
Protoplasmic Streaming ◽  
Intracellular Ca

Calcium is supposed to play an important role in the control of protoplasmic streaming in slime mold plasmodia. The motive force for protoplasmic streaming is generated by the interaction of actin and myosin. This contraction is supposed to be controlled by intracellular Ca-fluxes similar to the triggering system in skeleton muscle. The histochemical localisation of calcium however is problematic because of the possible diffusion artifacts especially in aquous media.To evaluate this problem calcium localisation was studied in small pieces of shock frozen (liquid propane at -189°C) plasmodial strands of Physarum polycephalum, which were further processed with 3 different methods: 1) freeze substitution in ethanol at -75°C, staining in 100% ethanol with 1% uranyl acetate, and embedding in styrene-methacrylate. For comparison the staining procedure was omitted in some preparations. 2)Freeze drying at about -95°C, followed by immersion with 100% ethanol containing 1% uranyl acetate, and embedding. 3) Freeze fracture, carbon coating and SEM investigation at temperatures below -100° C.

A pre-embedding double staining procedure used to observe the effect of fixation on the activity of intercellular adhesion molecule on T and B cells

1990 ◽  
Vol 48 (3) ◽  
pp. 378-379
Author(s):  
Jane E. Ramberg ◽  
Shigeto Tohma ◽  
Peter E. Lipsky
Keyword(s):  
B Cells ◽  
Adhesion Molecule ◽  
Colloidal Gold ◽  
Intercellular Adhesion ◽  
Intercellular Adhesion Molecule ◽  
Staining Procedure ◽  
Double Staining ◽  
T And B Cells ◽  
Microtiter Plates ◽  
Streptavidin Peroxidase

Intercellular adhesion molecule (ICAM-1) appears to be a ligand for LFA-1 dependent adhesion in T cell mediated cytotoxcity. It is found on cells of both hematopoietic and non-hematopoietic origin. While observing the activity of ICAM-1 on the surfaces of interacting T and B cells, we found that we could successfully carry out a pre-embedding double staining procedure utilizing both colloidal gold and peroxidase conjugated reagents.On 24-well microtiter plates, mitomycin-treated T4 cells were stimulated with 64.1 (anti-CD3) for one hour before the addition, in some instances, of B cells. Following a 12-48 hour incubation at 38°C, the cells were washed and then immunostained with a colloidal gold conjugated RFB-4 (anti-CD22); biotinylated R6.5 (anti-ICAM-1); followed by streptavidin/peroxidase. This method allowed us to observe two different antigens without concern about possible cross-reaction of reagents. Because we suspected ICAM-1 and R6.5 were sensitive to fixation, we tried varying concentrations of fresh paraformaldehyde before R6.5, after R6.5 and after streptavidin/peroxidase. All immunostaining and washing was done on ice with ice cold reagents.

Solvent-assisted osmium staining of butyl acrylate and ethylene-propylene-diene in a styrene acrylonitrile matrix

1993 ◽  
Vol 51 ◽  
pp. 900-901 ◽  
Author(s):  
Gudrun A. Hutchins
Keyword(s):  
Butyl Acrylate ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Staining Procedure ◽  
Reactive Site ◽  
Ethylene Propylene ◽  
Minimal Distortion ◽  
Styrene Acrylonitrile ◽  
Engineering Polymers ◽  
Effective Reaction

In order to optimize the toughening effect of elastomers in engineering polymers, it is necessary to characterize the size, morphology and dispersion of the specific elastomer within the polymer matrix. For unsaturated elastomers such as butadiene or isoprene, staining with osmium tetroxide is a well established procedure. The residual carbon-carbon double bond in these materials is the reactive site and forms a 1,2-dilato complex with the OsO4. Incorporation of osmium tetroxide into the elastomer not only produces sufficient contrast for TEM, but also crosslinks the elastomer sufficiently so that ultramicrotomy can be accomplished at room temperature with minimal distortion.Blends containing saturated elastomers such as butyl acrylate (BA) and ethylene propylene diene monomer (EPDM) cannot be stained directly with OsO4 because effective reaction sites such as C=C or -NH2 are not available in sufficient number. If additional reaction sites can be introduced selectively into the elastomer by a chemical reaction or the absorption of a solvent, a modified, two-step osmium staining procedure is possible.

Minimization of activated sludge production by chemically stimulated energy spilling

2000 ◽  
Vol 42 (12) ◽  
pp. 189-200 ◽  
Author(s):  
G.-H. Chen ◽  
H.-K. Mo ◽  
S. Saby ◽  
W.-k. Yip ◽  
Y. Liu
Keyword(s):  
Activated Sludge ◽  
Growth Yield ◽  
Dry Weight ◽  
Staining Technique ◽  
Microbial Culture ◽  
Dapi Staining ◽  
E Coli ◽  
Energy Spilling ◽  
Activated Sludge Processes ◽  
Sludge Production

Minimization of excess sludge production in activated sludge processes has been pursued around the world in order to meet stringent environmental regulations on sludge treatment and disposal. To achieve this goal, physical, chemical, and biological approaches have been proposed. In this paper, a chemical compound, 3,3′,4′,5-tetrachlorosalicylanilide (TCS) was tested for enhancing microbial energy spilling of the sludgeso as to minimize its growth. In order to examine this, an exploratory study was conducted using both batch and continuous activated sludge cultures. Batch experiments with these two cultures were carried out at different initial concentrations of TCS. It has been confirmed that an addition of TCS is effective in reducing the production of both the sludge cultures, particularly the continuous culture where the observed growth yield was reduced by around 70%, when the initial TCS concentration was 0.8 ppm. Meanwhile, the substrate removal activity of this culture was found not to be affected at this TCS concentration. To further evaluate the TCS effect, a pure microbial culture of E. coli was employed. Batch experiment results with this culture implied that TCS might be able to reduce the cell density of E. coli drastically when an initial TCS concentration was greater than 0.12 ppm. It was also found that TCS was not toxic to this type of bacteria. Microscopic examinations with a 4′, 6-diamidino-2-phenylindole (DAPI) staining technique revealed that TCS neither affected the cell division nor altered the cell size of E. coli. However, both the cell ATP content and the cell dry weight were reduced significantly with the addition of TCS.

Synthesis and Biological Evaluation of Novel Heterocyclic Imines Linked Coumarin- Thiazole Hybrids as Anticancer Agents

2019 ◽  
Vol 19 (4) ◽  
pp. 557-566 ◽  
Author(s):  
Nerella S. Goud ◽  
Mahammad S. Ghouse ◽  
Jatoth Vishnu ◽  
Jakkula Pranay ◽  
Ravi Alvala ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Membrane Potential ◽  
Fluorescence Spectroscopy ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Annexin V ◽  
Biological Evaluation ◽  
Dependent Manner ◽  
Dapi Staining ◽  
Dose Dependent

Background: Human Galectin-1, a protein of lectin family showing affinity towards β-galactosides has emerged as a critical regulator of tumor progression and metastasis, by modulating diverse biological events including homotypic cell aggregation, migration, apoptosis, angiogenesis and immune escape. Therefore, galectin-1 inhibitors might represent novel therapeutic agents for cancer. Methods: A new series of heterocyclic imines linked coumarin-thiazole hybrids (6a-6r) was synthesized and evaluated for its cytotoxic potential against a panel of six human cancer cell lines namely, lung (A549), prostate (DU-145), breast (MCF-7 & MDA-MB-231), colon (HCT-15 & HT-29) using MTT assay. Characteristic apoptotic assays like DAPI staining, cell cycle, annexin V and Mitochondrial membrane potential studies were performed for the most active compound. Furthermore, Gal-1 inhibition was confirmed by ELISA and fluorescence spectroscopy. Results: Among all, compound 6g 3-(2-(2-(pyridin-2-ylmethylene) hydrazineyl) thiazol-4-yl)-2H-chromen-2- one exhibited promising growth inhibition against HCT-15 colorectal cancer cells with an IC50 value of 1.28 ± 0.14 µM. The characteristic apoptotic morphological features like chromatin condensation, membrane blebbing and apoptotic body formation were clearly observed with compound 6g on HCT-15 cells using DAPI staining studies. Further, annexin V-FITC/PI assay confirmed effective early apoptosis induction by treatment with compound 6g. Loss of mitochondrial membrane potential and enhanced ROS generation were confirmed with JC-1 and DCFDA staining method, respectively by treatment with compound 6g, suggesting a possible mechanism for inducing apoptosis. Moreover, flow cytometric analysis revealed that compound 6g blocked G0/G1 phase of the cell cycle in a dose-dependent manner. Compound 6g effectively reduced the levels of Gal-1 protein in a dose-dependent manner. The binding constant (Ka) of 6g with Gal-1 was calculated from the intercept value which was observed as 1.9 x 107 M-1 by Fluorescence spectroscopy. Molecular docking studies showed strong interactions of compound 6g with Gal-1 protein. Conclusion: Our studies demonstrate the anticancer potential and Gal-1 inhibition of heterocyclic imines linked coumarin-thiazole hybrids.

scholarly journals Axon typing of rat muscle nerves using a double staining procedure for cholinesterase and carbonic anhydrase.

1991 ◽  
Vol 39 (12) ◽  
pp. 1617-1625 ◽  
Author(s):  
M J Szabolcs ◽  
A Windisch ◽  
R Koller ◽  
M Pensch
Keyword(s):  
Carbonic Anhydrase ◽  
Cross Sections ◽  
Sensory Nerve ◽  
Histological Section ◽  
Trapezius Muscle ◽  
Motor Units ◽  
Conventional Technique ◽  
Nerve Fibers ◽  
Staining Procedure ◽  
Muscle Nerve

We developed a method for detecting activity of axonal cholinesterase (CE) and carbonic anhydrase (CA)--markers for motor and sensory nerve fibers (NFs)--in the same histological section. To reach this goal, cross-sections of muscle nerves were sequentially incubated with the standard protocols for CE and CA histochemistry. A modified incubation medium was used for CA in which Co++ is replaced by Ni++. This avoids interference of the two histochemical reactions because Co++ binds unspecifically to the brown copper-ferroferricyanide complex representing CE activity, whereas Ni++ does not. Cross-sections of the trapezius muscle nerve containing efferent and afferent NFs in segregated fascicles showed that CE activity was confined to motor NFs. Axonal CA was detected solely in sensory NFs. The number of labeled motor and sensory NFs determined in serial cross-sections stained with either the new or the conventional technique was not significantly different. Morphometric analysis revealed that small unreactive NFs (diameter less than 5 microns) are afferent, medium-sized ones (5 microns less than d less than 7 microns) are unclassifiable, and large ones (d greater than 7 microns) are efferent. The heterogenous CE activity of thick (alpha) motor NFs is linked to the type of their motor units. "Fast" motor units contain CE reactive NFs; "slow" ones have CE negative neurites.

Evaluation of the Effect of Hyaluronic Acid–Based Biomaterial Enriched With Tenascin-C on the Behavior of the Neural Stem Cells

2021 ◽  
pp. 109158182110024
Author(s):  
Maryam Shahi ◽  
Daruosh Mohammadnejad ◽  
Mohammad Karimipour ◽  
Reza Rahbarghazi ◽  
Ali Abedelahi
Keyword(s):  
Stem Cells ◽  
Hyaluronic Acid ◽  
Neural Stem Cells ◽  
Mtt Assay ◽  
Dapi Staining ◽  
Biological Assays ◽  
Tenascin C ◽  
Electron Scanning Microscopy ◽  
Ethidium Bromide Staining ◽  
And Control

One of the most important natural extracellular constituents is hyaluronic acid (HA) with the potential to develop a highly organized microenvironment. In the present study, we enriched HA hydrogel with tenascin-C (TN-C) and examined the viability and survival of mouse neural stem cells (NSCs) using different biological assays. Following NSCs isolation and expansion, their phenotype was identified using flow cytometry analysis. Cell survival was measured using MTT assay and DAPI staining after exposure to various concentrations of 50, 100, 200, and 400 nM TN-C. Using acridine orange/ethidium bromide staining, we measured the number of live and necrotic cells after exposure to the combination of HA and TN-C. MTT assay revealed the highest NSCs viability rate in the group exposed to 100 nM TN-C compared to other groups, and a combination of 1% HA + 100 nM TN-C increased the viability of NSCs compared to the HA group after 24 hours. Electron scanning microscopy revealed the higher attachment of these cells to the HA + 100 nM TN-C substrate relative to the HA substrate. Epifluorescence imaging and DAPI staining of loaded cells on HA + 100 nM TN-C substrate significantly increased the number of NSCs per field over 72 hours compared to the HA group ( P < 0.05). Live and dead assay revealed that the number of live NSCs significantly increased in the HA + 100 TN-C group compared to HA and control groups. The enrichment of HA substrate with TN-C promoted viability and survival of NSCs and could be applied in neural tissue engineering approaches and regenerative medicine.

scholarly journals glp-3 Is Required for Mitosis and Meiosis in the Caenorhabditis elegans Germ Line

Genetics ◽  
1997 ◽  
Vol 145 (1) ◽  
pp. 111-121 ◽  
Author(s):  
Lisa C Kadyk ◽  
Eric J Lambie ◽  
Judith Kimble
Keyword(s):  
Caenorhabditis Elegans ◽  
Germ Cells ◽  
Germ Line ◽  
Stem Cell Population ◽  
Phenotypic Characterization ◽  
Loss Of Function ◽  
Dapi Staining ◽  
Cell Cycles ◽  
C Elegans ◽  
Mitosis And Meiosis

The germ line is the only tissue in Caenorhabditis elegans in which a stem cell population continues to divide mitotically throughout life; hence the cell cycles of the germ line and the soma are regulated differently. Here we report the genetic and phenotypic characterization of the glp-3 gene. In animals homozygous for each of five recessive loss-of-function alleles, germ cells in both hermaphrodites and males fail to progress through mitosis and meiosis, but somatic cells appear to divide normally. Germ cells in animals grown at 15° appear by DAPI staining to be uniformly arrested at the G2/M transition with &lt;20 germ cells per gonad on average, suggesting a checkpoint-mediated arrest. In contrast, germ cells in mutant animals grown at 25° frequently proliferate slowly during adulthood, eventually forming small germ lines with several hundred germ cells. Nevertheless, cells in these small germ lines never undergo meiosis. Double mutant analysis with mutations in other genes affecting germ cell proliferation supports the idea that glp-3 may encode a gene product that is required for the mitotic and meiotic cell cycles in the C. elegans germ line.

scholarly journals Integrated analysis of potential pathways by which aloe-emodin induces the apoptosis of colon cancer cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Dongxiao Jiang ◽  
Shufei Ding ◽  
Zhujun Mao ◽  
Liyan You ◽  
Yeping Ruan
Keyword(s):  
Colon Cancer ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Caspase 3 ◽  
Time Dependent ◽  
Integrated Analysis ◽  
Dependent Manner ◽  
Colon Cancer Cells ◽  
Dapi Staining ◽  
Aloe Emodin

Abstract Background Colon cancer is a malignant gastrointestinal tumour with high incidence, mortality and metastasis rates worldwide. Aloe-emodin is a monomer compound derived from hydroxyanthraquinone. Aloe-emodin produces a wide range of antitumour effects and is produced by rhubarb, aloe and other herbs. However, the mechanism by which aloe-emodin influences colon cancer is still unclear. We hope these findings will lead to the development of a new therapeutic strategy for the treatment of colon cancer in the clinic. Methods We identified the overlapping targets of aloe-emodin and colon cancer and performed protein–protein interaction (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. In addition, we selected apoptosis pathways for experimental verification with cell viability, cell proliferation, caspase-3 activity, DAPI staining, cell cycle and western blotting analyses to evaluate the apoptotic effect of aloe-emodin on colon cancer cells. Results The MTT assay and cell colony formation assay showed that aloe-emodin inhibited cell proliferation. DAPI staining confirmed that aloe-emodin induced apoptosis. Aloe-emodin upregulated the protein level of Bax and decreased the expression of Bcl-2, which activates caspase-3 and caspase-9. Furthermore, the protein expression level of cytochrome C increased in a time-dependent manner in the cytoplasm but decreased in a time-dependent manner in the mitochondria. Conclusion These results indicate that aloe-emodin may induce the apoptosis of human colon cancer cells through mitochondria-related pathways.

scholarly journals Three-dimensional in situ morphometrics of Mycobacterium tuberculosis infection within lesions by optical mesoscopy and novel acid-fast staining

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Robert J. Francis ◽  
Gillian Robb ◽  
Lee McCann ◽  
Bhagwati Khatri ◽  
James Keeble ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Three Dimensional ◽  
Large Field ◽  
Staining Procedure ◽  
3D Analysis ◽  
Mycobacterium Tuberculosis Infection ◽  
In Vivo Models ◽  
Novel Approach

AbstractTuberculosis (TB) preclinical testing relies on in vivo models including the mouse aerosol challenge model. The only method of determining colony morphometrics of TB infection in a tissue in situ is two-dimensional (2D) histopathology. 2D measurements consider heterogeneity within a single observable section but not above and below, which could contain critical information. Here we describe a novel approach, using optical clearing and a novel staining procedure with confocal microscopy and mesoscopy, for three-dimensional (3D) measurement of TB infection within lesions at sub-cellular resolution over a large field of view. We show TB morphometrics can be determined within lesion pathology, and differences in infection with different strains of Mycobacterium tuberculosis. Mesoscopy combined with the novel CUBIC Acid-Fast (CAF) staining procedure enables a quantitative approach to measure TB infection and allows 3D analysis of infection, providing a framework which could be used in the analysis of TB infection in situ.

Sign in / Sign up

Export Citation Format

Share Document