Analysis of apoptosis in zebrafish embryos by wholemount. We have worked out a cytochemical method, which is suitable for the specific demonstration of surface gag components. A total of 75 infertile men with varicocele and 40 fertile men controls were included in this study. The method consists of a glutaraldehyde prefixation, an acridine orange block staining for 48 h and an oso4 postfixation for some hours.
Liver apoptosis can be observed after neomycin treatment. Being cellpermeable, it interacts with dna and rna by intercalation or electrostatic attractions respectively. Acridine orange staining and visualization in zebrafish to. Acridine orange propidium iodide double staining for apoptotic and necrotic cell determination. The protocol described herein uses an antibody that specifically binds the cleaved, heterotetrameric form of caspase 3, but not the inactive caspase 3 dimer. The influence that changes in proton distribution have on the fluorescence of acridine orange was examined using negatively charged liposomes. Acridine orange staining has been shown to be highly selective for apoptotic cells in drosophila. Live zebrafish embryos were stained with the vital dye acridine orange ao in. Provide content correction we continue to work to improve your shopping experience and your feedback regarding this content is very important to us. Im using zebrafish as invivo models to study apoptosis induced by metalcompounds. With the nucleic acid binding dyes acridine orange ao and propidium iodide pi you can accurately determine cell viability. It emits red, however, upon interacting with ssdna.
Dec 20, 20 wholemount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. Acridine orange staining and visualization in zebrafish to study apoptosis. Acridine orange ao is a vital dye often used as a marker of apoptotic cells in zebrafish. Using the zebrafish danio retio model, we asked whether apoptosis or.
In the present study we standardised a protocol in order to use acridine orange to detect rotavirus ds rna in polyacrylamide gels and compared it to silver and ethidium bromide staining. Tunel or hoechst if you have nuclei in your sample zebra fish. In 1980, a study involved the comparing acridine orange staining with blind subcultures for the detection of positive blood cultures was done by mccarthy and senne. Acridine orange is a cellpermeable fluorescent dye that binds to nucleic acids, resulting in an altered spectral emission. At low ph inside the organelles, it will emit an orange fluorescence peak at 590 nm. Acridine yellow, also known as acridine yellow g, acridine yellow h107, basic yellow k, and 3,6diamino2,7dimethylacridine, is a yellow dye with strong bluishgreen fluorescence. Within acidic vesicles, acridine orange becomes protonated and trapped within the organelle. General staining and embryo preparation for microscopy protocol. Using acridine orange to measure cell death in ethanol treated. Acridine orange stain principle, procedure and result interpretation. Apr 07, 2010 acridine orange ao is a vital dye often used as a marker of apoptotic cells in zebrafish.
Please use the form below to provide feedback related to the content on this product. Acridine orange ao stains dsdna green 525 nm and rna or single stranded dna red 650 nm. Role of programmed cell death in defining zebrafish development. Protocol acridine orange ethidium bromide aoeb staining to detect apoptosis. One exception that we are aware of is gelgreen, which is more sensitive to light exposure than most of our other fluorescent dyes. Acridine orange definition of acridine orange by medical. A rapid apoptosis assay measuring relative acridine orange fluorescence in zebrafish embryos.
Druginduced apoptosis visualized by acridine orange staining. If this occurs, the gelgreen will no longer work for gel staining. At neutral phs, acridine orange is a hydrophobic green fluorescent molecule. Low concentrations of acridine orange and propidium iodide aopi were used to visualize living and dead islet cells simultaneously. Incubate alive embryos at room temperature with acridine orange 1x during 30 minutes in e3 medium. Acridine orange staining of virus infected host cells to monitor proliferation of viralinfection full paper. Electron micrographs of apoptotic cell death within the zebrafish embryo at shield. It is used as a nucleic acidselective fluorescent cationic dye useful for cell cycle determination. Histological methods protocols zfin community wiki.
We offer a highly purified form of acridine orange while most of the. Rna electrostatically bound ao fluoresces red 630nm. Acridine orange stain definition of acridine orange stain. Acridine orange staining is a sensitive, rapid and reliable method for detecting bacteria in blood cultures early during incubation and can be substituted for blind subcultures. M, we demonstrated that all eleven bands of virus ds rna emitted a weak. Aoeb working solution 1 add ao and eb to pbs and mix well. Cells are automatically stained by the two fluorophores acridine orange and dapi staining total cells and dead cells, respectively. Acridine orange has also been used as a lysosomal dye. When ao intercalates into dsdna it emits green fluorescence upon excitation at.
The dye is membranepermeant and its nucleic acid binding property has been used for cellcycle studies. Acridine orange is a lysotropic dye that accumulates in acidic organelles in a phdependent manner. Using the vital dye acridine orange to detect dying cells. Acridine orange is a fluorescent dye which will bind to the nucleic acid of bacteria and other cells.
Similar to other animals, in zebrafish embryos, programmed cell death. Acridine orange staining of virus infected host cells to. We demonstrated that the simplest and best condition was. Acridine orange not in an acidic compartment will emit as green. Aoeb or aopi for viability mario roderer 602 reagents. The protocol described herein uses an antibody that specifically binds. Cell apoptosis acridine orange detection kit kit genscript. Acridine orange 10 mgml in water thermo fisher scientific. Acridine orange sigma a6014 is prepared at 1mgml 100x in milliq water and stored at 20c light protected. Acridine orange staining of the mammalian fibroblast cell coat. Use of acridine orange staining for the detection of. The wholemount analysis provides spatial information in regard to tissue specificity. Protocol for acridine orange staining after dna denaturation acridine orange ao is a metachromatic dye which differentially stains doublestranded ds and singlestranded ss nucleic acids.
Acridine orange, 10 mgml in water high purity ao biotium. Semen analysis and sperm dna damage expressed as the dna fragmentation index using acridine orange staining and chromatin condensation test by flow cytometry were assessed before and 6 months after varicocelectomy. Acridine orange stain definition of acridine orange. It crosses into lysosomes and other acidic compartments and becomes protonated. Visualization of dead cells with acridine orange protocols. Acridine orange, which is a metachromatic intercalator sensitive to dna conformation, has been widely used to detect apoptosis in wildtype, mutant, and drugtreated zebrafish brand et al. Apoptosis analysis in zebrafish embryos was done with the help of experimental protocol for acridine orange ao staining described by tucker and lardelli 2007. Acridine orange staining and visualization in zebrafish to study. Propidium iodide can only enter dead cells with poor membrane intergrity, so it will stain all dead nucleated cells to generate red fluorescence. Amarantemendes, deborah finucane, thomas brunner, ella bossywetzel, and douglas r. Our results indicate that at least two mechanisms are involved.
It is a differential staining of nucleic acids including dna and rna. Use of acridine orange staining for the detection of rotavirus rna in polyacrylamide gels. Vectacell acridine orange is a fluorescent dye that can be used to stain acidic organelles, such as lysosomes, autosomes or yeast vacuoles. This cellpermeant cellular stain can be utilized in conjunction with a number of other staining techniques and fluorogenic substrates including the magic red line of fluorogenic protease substrates. Acridine orange stain principle, procedure and result. A rapid apoptosis assay measuring relative acridine orange. When acridine orange staining was performed before electrophoresis at a dye concentration of 21. Recently it was shown that ao can also be used in live imaging to observe cell death in the zebrafish embryonic spinal cord in a model of inherited cognitive disease related to mutant tau. The tunel assay was adapted from established protocols gavrieli et. Additional wholemount techniques to identify apoptotic cells in zebrafish embryos include the tunel assay 8 and acridine orange ao staining 9. Rapid assessment of islet viability with acridine orange and.
Acridine orange is used for fluorescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status. Diagnosis of malaria by acridine orange fluorescent. Using acridine orange to measure cell death in ethanol. This unique characteristic makes acridine orange useful for cellcycle studies. Acridine orange ao exhibits metachromatic fluorescence that is. Shop online for a wide selection of bd acridine orange stain for detecting microorganisms in direct smears by fluorescence. Acridine orange is a fluorescent pigment, the detection wavelength of 488 nm excitation filter. Compared to acridine orange staining, which can identify apoptotic cells in. Clearing and staining for larval fish cartilage and bone. Vectacell acridine orange for live cell imaging of organelles. When bound to dna, it is very similar spectrally to fluorescein, with an excitation maximum at 502 nm and an emission maximum at 525 nm green. Acridine orange staining solution for lysosome and dna staining.
Acridine orange is an intercalating dye that can permeate both live and dead cells. The vital dyes acridine orange and dansylcadaverine mdc are commonly used to study autophagy. A simple, rapid method for estimating the viability of isolated islets of langerhans with fluorescent dyes is described. Ao will stain all nucleated cells to generate green fluorescence.
Wholemount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. Reagents for analyzing autophagy autophagy sigmaaldrich. Im using zebrafish as invivo models to study apoptosis induced by metal compounds. To study this interesting possibility, the zebrafish danio rerio embryo is.
Wash 3 times during 10 minutes shacking is optional. Acridine orange staining solution for lysosome and dna. If gelgreen is exposed to ambient light for a prolonged period of time days to weeks, its color will change from dark orange to brick red. Protocol acridine orangeethidium bromide aoeb staining to detect apoptosis. Acridine orange staining procedure acridine orange ao is a nucleic acid selective metachromatic stain useful for cell cycle determination.
Developmental apoptosis mediates entry and positioning of. Fluorescent acridine orange microscopical examination of capillary centrifuged blood quantitative buffy coat qbc analysis and giemsa stained thick blood smears gts were compared for diagnosis of malaria in blood specimens from adults living in malaria transmission areas of the states of bolivar and amazonas in southeastern and south venezuela, respectively. With cell dna and rnabinding capacity gaps exist, it may issue a different color fluorescence, and dna binding of less green fluorescence, and the amount of rnabinding orange or orange red fluorescence. Ao interacts with dna and rna by intercalation or electrostatic attraction respectively.
Aopistained islets can be divided into three distinct groups. Untreated zebrafish a, 4dayold zebrafish were treated with 1. Staining protocol if cells used in tissue culture are attached, then. Acridine orange staining technique a quick diagnostic for babesia gibsoni infection. It is recommended for use for the detection of microorganisms in direct smears. Acridine orange staining of virus infected host cells to monitor proliferation of viralinfection full paper introduction acridine orange ao interacts with dna and rna by intercalation and electrostatic attraction, respectively1. When ao intercalates into dsdna, it releases green fluorescence upon excitation at 480490 nm. In histology, it is used as a fluorescent stain, and as a fluorescent probe for noninvasive measurements of cytoplasmic ph changes in whole cells.
Can acridine orange be used to stain dna and rna in different fluorescent colors in sections as well as in smears of cells. Although this method may label many of the same apoptotic cells. Ao is cell permeable and can interact with nucleic acid. Acridine orange for microscopical staining loba chemie. Semen analysis and sperm dna damage expressed as the dna fragmentation index using acridine orange staining and chromatin condensation test by flow cytometry were. Acridine orange is better than gram stain in cases with low amounts of organisms. This cellpermeant cellular stain can be utilized in conjunction with a number of other staining techniques and fluorogenic substrates including the. The protonated dye stacks and stacked acridine orange emits in the red range.
After 60 minutes of staining embryos were washed three times in e3 media. The use of acridine orange in clinical applications has become widely accepted. A simple wholemount staining protocol for bone and or cartilage in adults and larvae. Similar to other animals, in zebrafish embryos, programmed cell death maintains the homeostasis of organ development, removes surplus cells to form. Acridine orange ao is a metachromatic dye which differentially stains doublestranded ds and singlestranded ss nucleic acids. Acridine orangeethidium bromide aoeb staining to detect. A quick and easy method to determine the number of viable, apoptotic and necrotic cells in a culture. Millipore was used following the protocol suggested by the provider. Rapid assessment of islet viability with acridine orange. For optimal endosome visualization use a blue light excitation 475 nm. When ao intercalates into dsdna it emits green fluorescence upon excitation at 480490 nm. The fluorochrome inserts into nucleic acid and fluoresces orange when attached to bacteria or fungi. Samples stained with acridine orange are viewed on a fluorescent microscope. Phosphatidylserine receptor is required for the engulfment of dead.
Group a islets fluoresce green, contain insulin, and have normal. The interaction of acridine orange with target molecules i. Apoptotic cells stain heavily with ao due to the increased binding of. Dna and rna is by intercalation or electrostatic attraction, respectively. Acridine orange propidium iodide double staining staining. Advantages of acridine orange staining include the speed and ease of the staining. Zebrafish module mbl embryology course 2010 june 29th july 3rd manual modified from original version written by marnie halpern and josh gamse, with many contributions from other instructors and tas over the years.
Advanced cell image analysis for cell biology research and development by the use of a broad range of plugnplay assays. Acridine orange staining was performed, as described previously furutaniseiki et al. Acridine orange is a cellpermeant nucleic acid binding dye that emits green fluorescence when bound to dsdna and red fluorescence when bound to ssdna or rna. In order to test the best protocol for acridine orange staining, gels were loaded with ng of rotavirus ds rna per well, throughout the experiments, unless otherwise stated.
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