Clearing and staining for larval fish cartilage and bone. Acridine orange staining has been shown to be highly selective for apoptotic cells in drosophila. Staining protocol if cells used in tissue culture are attached, then. Gentle fixation by freeze substitution gives excellent histological results with zebrafish embryos. It crosses into lysosomes and other acidic compartments and becomes protonated.
The tunel assay was adapted from established protocols gavrieli et. Acridine orange staining live zebrafish embryos were stained with the vital dye acridine orange ao in order to determine the amount of apoptotic cells per embryo. 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. We perform image processing and analysis to generate a focused image from an image stack and then use the object masking capabilities of gen5 to count the number of green ao positive cells per embryo as well as determine the total amount of ao fluorescence per embryo. 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. The protonated dye stacks and stacked acridine orange emits in the red range. Determination of micronucleus frequency by acridine orange. Apoptotic cells stain heavily with ao due to the increased binding of. Caspasemediated apoptosis induction in zebrafish cerebellar purkinje.
Liver apoptosis can be observed after neomycin treatment. Acridine orange as a cytotopochemical reagent for nucleic acids dna and rna is discussed in detail. We demonstrated that the simplest and best condition was. Acridine orange stain principle, procedure and result. This cellpermeant cellular stain can be utilized in conjunction with a number of other staining techniques and fluorogenic substrates including the. Druginduced apoptosis visualized by acridine orange staining.
Rna electrostatically bound ao fluoresces red 630nm. Similar to other animals, in zebrafish embryos, programmed cell death. Acridine orange staining and visualization in zebrafish to study apoptosis. Analysis of apoptosis in zebrafish embryos by wholemount. Analyzing apoptosis has been an integral component of many biological studies. Acridine orange ao stains dsdna green 525 nm and rna or single stranded dna red 650 nm. Cell permeability, safety, and sensitivity of ethidium bromide alternatives. A quick and easy method to determine the number of viable, apoptotic and necrotic cells in a culture. The interaction of acridine orange with target molecules i. Compared to acridine orange staining, which can identify apoptotic cells in. The dye is membranepermeant and its nucleic acid binding property has been used for cellcycle studies.
Acridine orange staining solution for lysosome and dna. Amarantemendes, deborah finucane, thomas brunner, ella bossywetzel, and douglas r. Millipore was used following the protocol suggested by the provider. Acridine orange is better than gram stain in cases with low amounts of organisms. Photoconversion of fluorescently labeled profiles for em.
Incubate alive embryos at room temperature with acridine orange 1x during 30 minutes in e3 medium. Developmental apoptosis mediates entry and positioning of. This has especially been applied i to cytodiagnosis of cancer. When acridine orange associates with rna, the maximum excitation shifts to 460 nm, and the maximum emission shif. 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. 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. Samples stained with acridine orange are viewed on a fluorescent microscope. 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. 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. Aoeb or aopi for viability mario roderer 602 reagents.
After 60 minutes of staining embryos were washed three times in e3 media. Flow cytometry reticulocyte counting using acridine orange. M, we demonstrated that all eleven bands of virus ds rna emitted a weak. A rapid apoptosis assay measuring relative acridine orange fluorescence in zebrafish embryos. We have used all three methods to identify apoptotic cells in zebrafish embryos 4,6,10. Acridine orange staining is nice for a quick and dirty test of cell death, but tunel is the gold standard and still easy to do wholemount or on sections. When ao intercalates into dsdna it emits green fluorescence upon excitation at 480490 nm. Tunel or hoechst if you have nuclei in your sample zebra fish. Faq home staining methods, histochemistry acridine orange method for dna and rna question. Acridine orange stain article about acridine orange stain.
Acridine orange staining was performed, as described previously furutaniseiki et al. Using the zebrafish danio retio model, we asked whether apoptosis or autophagy was a default. Acridine orange sigma a6014 is prepared at 1mgml 100x in milliq water and stored at 20c light protected. February 1, 2017 comparison of nucleic acid gel stains. It is used as a nucleic acidselective fluorescent cationic dye useful for cell cycle determination. 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. To study this interesting possibility, the zebrafish danio rerio embryo is. Acridine orange stain definition of acridine orange. Ao will stain all nucleated cells to generate green fluorescence.
Acridine orange staining of the mammalian fibroblast cell coat. For optimal endosome visualization use a blue light excitation 475 nm. At low ph inside the organelles, it will emit an orange fluorescence peak at 590 nm. Im using zebrafish as invivo models to study apoptosis induced by metal compounds. This unique characteristic makes acridine orange useful for cellcycle studies. Zebrafish embryos were treated with ethanol during the first 24 hours of development and the effects of ethanol treatment on cell death was assessed using acridine orange staining. Acridine orange staining and visualization in zebrafish to study.
Using acridine orange to measure cell death in ethanol. Acridine orange is a cellpermeable fluorescent dye that binds to nucleic acids, resulting in an altered spectral emission. Cell apoptosis acridine orange detection kit kit genscript. Acridine orange for microscopical staining loba chemie. A simple wholemount staining protocol for bone and or cartilage in adults and larvae. 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. Apr 07, 2010 acridine orange ao is a vital dye often used as a marker of apoptotic cells in zebrafish. In this study we use acridine orange ao staining combined with gen5 microplate reader and imager oftware to determine the amount of apoptotic cell death in zebrafish embryos after 24 hours of ethanol exposure. Acridine orange staining of virus infected host cells to monitor proliferation of viralinfection full paper. However, currently available methods for quantifying apoptosis have various limitations including multiple, sometimes celldamaging steps, the inability to quantify live, necrotic and apoptotic cells at the same time, and nonspecific detection i.
A total of 75 infertile men with varicocele and 40 fertile men controls were included in this study. Aoeb working solution 1 add ao and eb to pbs and mix well. Acridine orange is a fluorescent pigment, the detection wavelength of 488 nm excitation filter. Shop online for a wide selection of bd acridine orange stain for detecting microorganisms in direct smears by fluorescence. At neutral phs, acridine orange is a hydrophobic green fluorescent molecule.
The wholemount analysis provides spatial information in regard to tissue specificity. Additional wholemount techniques to identify apoptotic cells in zebrafish embryos include the tunel assay 8 and acridine orange ao staining 9. Although this method may label many of the same apoptotic cells. Using the vital dye acridine orange to detect dying cells in.
Within acidic vesicles, acridine orange becomes protonated and trapped within the organelle. Live zebrafish embryos were stained with the vital dye acridine orange ao in. Wash 3 times during 10 minutes shacking is optional. This study aimed to validate a laboratory protocol for flow. Advantages of acridine orange staining include the speed and ease of the staining. Untreated zebrafish a, 4dayold zebrafish were treated with 1. A simple technique for quantifying apoptosis in 96well. Propidium iodide can only enter dead cells with poor membrane intergrity, so it will stain all dead nucleated cells to generate red fluorescence. When ao intercalates into dsdna, it releases green fluorescence upon excitation at 480490 nm.
Acridine orange has also been used as a lysosomal dye. We have worked out a cytochemical method, which is suitable for the specific demonstration of surface gag components. Acridine orange stain definition of acridine orange stain. Acridine orange staining of virus infected host cells to. Acridine orange stains dna with green fluorescence and rna and acidic organelles. 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 is a lysotropic dye that accumulates in acidic organelles in a phdependent manner. 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. The method consists of a glutaraldehyde prefixation, an acridine orange block staining for 48 h and an oso4 postfixation for some hours.
When acridine orange staining was performed before electrophoresis at a dye concentration of 21. When bound to dna, it is very similar spectrally to fluorescein, with a maximum excitation at 502 nm and 525 nm. Im using zebrafish as invivo models to study apoptosis induced by metalcompounds. Wholemount staining of biotindextran injected embryos. Acridine orange staining solution for lysosome and dna staining.
Using the vital dye acridine orange to detect dying cells. Can acridine orange be used to stain dna and rna in different fluorescent colors in sections as well as in smears of cells. A study involving mccarthy and senne comparing acridine orange staining with blind subcultures for the detection of positive blood cultures showed that the acridine orange is a simple, inexpensive, rapid staining procedure that appeared to be more sensitive than the gram stain for detecting microorganisms. 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. When ao intercalates into dsdna it emits green fluorescence upon excitation at. Being cellpermeable, it interacts with dna and rna by intercalation or electrostatic attractions, respectively. Acridine orange not in an acidic compartment will emit as green. Acridine orange propidium iodide double staining for apoptotic and necrotic cell determination. Thermo scientific remel acridine orange is a fluorescent stain for detection of bacteria in body fluids. Acridine orange staining and visualization in zebrafish to. Dna and rna is by intercalation or electrostatic attraction, respectively. Protocol acridine orange ethidium bromide aoeb staining to detect apoptosis.
Acridine orange stain article about acridine orange. Acridine orange definition of acridine orange by medical. Acridine orange ao exhibits metachromatic fluorescence that is. Acridine orange is an intercalating dye that can permeate both live and dead cells. Ao interacts with dna and rna by intercalation or electrostatic attraction respectively. Wholemount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos.
Acridine orangeethidium bromide aoeb staining to detect. Use of acridine orange staining for the detection of. 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. With the nucleic acid binding dyes acridine orange ao and propidium iodide pi you can accurately determine cell viability.
Similar to other animals, in zebrafish embryos, programmed cell death maintains the homeostasis of organ development, removes surplus cells to form. Vectacell acridine orange is a fluorescent dye that can be used to stain acidic organelles, such as lysosomes, autosomes or yeast vacuoles. Vectacell acridine orange for live cell imaging of organelles. The method consists of a glutaraldehyde prefixation, an acridine orange blockstaining for 48 h and an oso4 postfixation for some hours. We perform image processing and analysis to generate a focused image from an image. Acridine orange staining procedure acridine orange ao is a nucleic acid selective metachromatic stain useful for cell cycle determination. Semen analysis and sperm dna damage expressed as the dna fragmentation index using acridine orange staining and chromatin condensation test by flow cytometry were. We found that exposure of embryos to ethanol results in a dosedependent increase in cell death overall in the embryo.
Acridine orange stain principle, procedure and result interpretation. While the casp3 protocol gives robust, reliable staining of apoptotic cells in embryos from the 4cell stage through 32 hpf, it has limited application beyond 32 hpf due to the inability of the. Acridine orange propidium iodide double staining staining. It is used as a nucleic acidselective fluorescent dye, that has cationic properties useful for cell cycle determination. Role of programmed cell death in defining zebrafish development.
Visualization of dead cells with acridine orange protocols. It is a differential staining of nucleic acids including dna and rna. Acridine orange ao is a vital dye often used as a marker of apoptotic cells in zebrafish. Use of acridine orange staining for the detection of rotavirus rna in polyacrylamide gels.
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. When beijerinck enriched for nitrogen fixers, he inoculated soil into two types of liquid media. Using acridine orange to measure cell death in ethanol treated. The vital dyes acridine orange and dansylcadaverine mdc are commonly used to study autophagy. The protocol described herein uses an antibody that specifically binds. A rapid apoptosis assay measuring relative acridine orange.
General staining and embryo preparation for microscopy protocol. Acridine orange is used for fluorescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status. The proposed ars vital staining protocol can be combined with gfp fluorescence associated with skeletal tissues and thus represents a powerful tool for in vivo monitoring of mineralized structures. Determination of micronucleus frequency by acridine orange fluorescent staining in peripheral blood reticulocytes of mice treated topically with different lubricant oils and cyclophosphamide c. We offer a highly purified form of acridine orange while most of the. It emits red, however, upon interacting with ssdna.
Reagents for analyzing autophagy autophagy sigmaaldrich. Protocol acridine orangeethidium bromide aoeb staining to detect apoptosis. Histological methods protocols zfin community wiki. The fluorochrome inserts into nucleic acid and fluoresces orange when attached to bacteria or fungi.
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