Calcein marking, a promising new tool for hatchery product. Adult zebrafish injury models to study the effects of. In most cases a staining time of 5 to 15 min is adequate. Compared with alizarin red staining used by fleming et al. Briefly, mice were intraperitoneally injected with 20 mgkg of calcein sigmaaldrich or 30 mgkg of alizarin sigmaaldrich in. Examination of stained, washed cells with confocal imaging allowed for increased sensitivity and higher spatial resolution of the fluorescence signal, especially for tracking production of new coccoliths. Once inside the cells, calcein am is hydrolyzed by endogenous esterase into the highly negatively charged green fluorescent calcein, which is retained in the cytoplasm. In live cells, nonfluorescent calcein am is converted to greenfluorescent calcein, after acetoxymethyl ester hydrolysis by intracellular esterases. Pdf visualizing normal and defective bone development in. Labelling cells with calcein am allows for them to be quantitated using a fluorescent plate reader and is useful for various in vitro assays, including chemotaxis assays. Calcein am is a nonfluorescent, hydrophobic compound that easily permeates intact, live cells. Calcein information calcein staining of fish to be released into the wild is not yet approved for general use. Calcein am has the ability to penetrate intact cornea, revealing cell viability, morphology and organization of living cornea.
In vitro assay of mineralizedtissue formation on titanium. With respect to the effect of bmp2 on skeletal formation, we found that ectopic expression of bmp2 in the notochord. Briefly, mice were intraperitoneally injected with 20 mgkg of calcein sigmaaldrich or 30 mgkg of alizarin sigmaaldrich in a 2% sodium bicarbonate solution. Revisiting in vivo staining with alizarin red s a valuable approach. Hardcopies of the 4th edition of the zebrafish book can be obtained for a nominal fee from the zebrafish international resource center, 5274 university of oregon, eugene, or 97403 usa. This is where zebrafish researchers can share experimental protocols and tips with the rest of the research community. Its bright green fluorescence can be monitored at exem494nm 520 nm figure 5. This technique can be used to determine, with the presence or absence of a fluorescent signal, whether specific genetic elements exist in a sample. Calcein am, calcein violet am, and calcein blue am labeling dyes cross the cell membrane easily, selectively labeling live cells for analysis by flow cytometry or fluorescent microscopy. Calcein am is membranepermeant and thus can be introduced into cells via incubation. Zebrafish embryology and cartilage staining protocols for. Dual staining methods using aopi, cfdapi, calcein ampi, hoechstpi, hoechstdraq7, and draq5dapi that enumerate viable and nonviable cells were also performed.
Zebrafish embryos were exposed to different zncl 2 concentrations and analyzed by icpms. Jan 17, 2018 clearing and staining for larval fish cartilage and bone. Zebrafish embryology and cartilage staining protocols for high school students article in zebrafish 62. We all know only live cells can be stained by calcein am, but after the staining, the live cells.
Using this novel method, we found that caion implantation on titanium inhibited mineralizednodule formation in vitro, while the immobilization of bisphosphonate on caionimpregnated titanium stimulated mineralizednodule formation. Histochemical detection of alkaline phosphatase alp activity revealed that abnormal segmentation of notochord induced by ra during vertebral development in zebrafish. Celltrace calcein green am is a cellpermeant dye that can be used to determine cell viability in most eukaryotic cells. Mar 15, 2011 research highlights we study the suitability of calcein as in situ growth marker in burrowing bivalves. The zebrafish is an ideal model that could link preclinical toxicity screening with the drug development pipeline. Zebrafish models for ectopic mineralization disorders. Calcein staining of 6 dpf embryos showing staining of the. Protocols are organized into sections corresponding to the chapters of the zebrafish book, 5th edition 4th edition online. Marking success increases with higher calcein concentrations and decreases with age. Frontiers calcein staining as a tool to investigate. Jan 19, 2016 in addition, the fluorescence spectrum of calcein is similar to that obtained with fish tissue autofluorescence.
In the first part of the study, the follicles viability was assessed by trypan. The kitchen cabinets you referred to were likely dyed, not stained. On the pathway of mineral deposition in larval zebra. Due to considerations on the bone developmental progress, early observation with calcein staining would be insufficiently clear to show the mineralization. Forgive me if i make obvious statements or odd comments as im not familar working with this kind of veneer andor staining process. In live cells the nonfluorescent celltrace calcein green am is converted to a greenfluorescent calcein after intracellular esterases remove the acetoxymethyl am esters. The zebrafish osteoporosis model could be induced by gluco. No detrimental effects are detected when using calcein concentrations. The skeletal anomalies were evaluated to confocal microscope after staining with calcein solution and rhodzin tm3,am. Visualizing normal and defective bone development in zebrafish embryos using the fluorescent chromophore calcein article pdf available in developmental biology 2382. The fluorescent chromophore, calcein c 30 h 26 n 2 o, specifically binds to calcium, fluorescently staining the calcified skeletal structures in living zebrafish larvae and juveniles fig. Viability and cytotoxicity assay reagentssection 15.
Cellular viability calcein propidium iodide weve performed this assay on the following cell types. The bluestar flashlight and the stereo microscope fluorescence adapter system are not endorsed by u. Revisiting in vivo staining with alizarin red s a valuable. We test how calcein concentration affects marking success and bivalve performance. Icts advanced calcein am cell viability kit combines calcein am with 7aad to allow for easy and simultaneous labeling of live, membrane compromised, and dead cells within a single sample. Fish is a molecular technique that is often used to identify and enumerate specific microbial groups. Calcein blue am is useful for viability measurements in combination with our sytox green nucleic acid stain see below and other green or redfluorescent probes. Calcein, also known as fluorexon, fluorescein complex, is a fluorescent dye with excitation and emission wavelengths of 495515 nm, respectively, and has the appearance of orange crystals.
Evaluation of zebrafish danio rerio ovarian follicle viability by simultaneous staining with fluorescein diacetate and propidium iodide article pdf available in cryo letters 296. Calcein is a green fluorescent molecule whose acetomethoxy derivative calcein am can be transported across cell membranes and therefore used to label live cells. Calcein selfquenches at concentrations above 70mm and is commonly used as an indicator of lipid vesicle leakage. Mar 12, 2014 we describe a new, highly sensitive semiquantitative method for rapid measurement of in vitro mineralization using calcein. Screening in larval zebrafish reveals tissuespecific distribution of. Clearing and staining for larval fish cartilage and bone. The color of the end product of the dab reaction may be altered by adding a heavy metal such as nickel or cobalt to the staining solution. Analysis of apoptosis in zebrafish embryos by wholemount. Calcein am staining solution 2x prepare a calcein am staining solution by adding 2 l of cellbased assay calcein am item no. On the pathway of mineral deposition in larval zebrafish.
In this research, we screened the f3 generation embryos and larvae derived from enu mutagenized founders. It should, however, be noted that ars concentrations used here were much lower 0. Quantitative analysis of activatedcaspase 3 can be performed utilizing software programs that measure fluorescence intensity of documented images. The skeletal anomalies were evaluated to confocal microscope after staining with calcein solution and rhodzintm3,am.
Moreover, two structurally similar chemotherapeutic compounds, doxorubicin and epirubicin, were observed to have. Apr 29, 2016 the increase of heavy metals in the environment involves a high exposure of aquatic organisms to these pollutants. The hydrolysis of calcein am by intracellular esterases. A guide for the laboratory use of zebrafish danio rerio. The objective of the present study is to assess viability tests and to evaluate follicle ovarian tissue quality after freezingthawing procedures. Stain has pigments that lodge in the crevasses of the wood grain, pores, bad sanding. To determine whether calcein staining could also be used to detect abnormal. The nonfluorescent calcein am dye is hydrolyzed by cellular esterases to give calcein which is fluorescent and is retained in the cytoplasm. The enzymatic conversion of the virtually nonfluorescent cellpermeant calcein violet am to the intensely fluorescent. Substantial information gathered from developmental and genetic research, together with nearcompletion of the zebrafish genome project, has placed zebrafish in an attractive position for use as a toxicological model.
Fix small pieces of tissue at room temperature for 2 h, then hold overnight at 4 0 c. Calcein staining of fish to be released into the wild is not yet approved for general use. In this paper, we outline our protocol for embedding, serial sectioning, staining and visualizing zebrafish. Summary zebrafish and medaka have become popular models for. Together, these results clearly demonstrated the sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants that have bone structure defects. Zebrafish embryos were exposed to different zncl2 concentrations and analyzed by icpms. Hardcopies of the 4th edition of the zebrafish book can be obtained for a nominal fee from the zebrafish international resource center, 5274. Dye will add color and really highlight the grain without muddying it up like a stain will sometimes do. For the fish general purpose reagents protocols provided in chapter 2 and chapter 3, the starting material is a spread of interphase or metaphase chromosomes fixed to a glass microscope slide. Upper and lower panels are dorsal and lateral views, respectively. At earlier stages, the terminal somite boundaries can be used to identify segments corresponding to pu2 and pu3. Incubate, using a shaker if possible, until sections show color. Calcein staining is a fluorescent dye used for detecting calcium as an index of mineralization.
The only other conclusion i can think of is the actual dna of the tree. Visualizing normal and defective bone development in. In conclusion, we developed a method of evaluating mineralizedtissue formation on titanium using calcein blue. Toxic effects of zinc chloride on the bone development in. Zebrafish zf, danio rerio has emerged as an important and popular model. Fluorescence analysis of the calcein bound to the calcium phosphate hydroxyapatite allows direct quantitation of extracellular matrix mineral content in monolayer cultures of boneforming cells such as primary osteoblasts or osteosarcoma cells. Live fluorescent staining platform for drugscreening and. Screening in larval zebrafish reveals tissuespecific. Here, we developed a novel methodology to directly evaluate the bone mass. Calcein am is used to detect live cells, fluorescing green, while 7aad is used to detect necrotic or late stage apoptotic cells, fluorescing red. Calcein am or calcein acetoxymethyl ester is a hydrophobic compound which passes easily through cell membrane into live cells and is used for cell viability assays. Visualizing normal and defective bone development in zebrafish. Oct 15, 2001 visualizing normal and defective bone development in zebrafish embryos using the fluorescent chromophore calcein.
Zebrafish could enhance preclinical drug screening by its strategic placement. The calcein am staining solution will be stable for one hour at room temperature. This allows analysis of bone in live zebrafish with high sensitivity, though it. Cellstain double staining kit combines calcein am used for fluorescentstaining the living cells and propidium iodide used for a fluorescent staining of the dead cells for simultaneous staining of the living and the dead cells. In vivo calcein labeling and calculation of bone formation. Thus, alternatives to calcein for zebrafish skeletal staining are desirable. Compared with alcian blue staining, we found that calcein staining indeed. Vital staining of fish bone is accomplished with two alizarin variants. By comparison with alcian blue staining, we showed that calcein staining indeed labels calci. Zebrafish danio rerio has been a prominent model vertebrate in a variety of biological disciplines. Here we applied calcein staining to examine skeletal development in red sea bream larvae. Additionally, we have found that ao staining diminishes after fixation with pfa, so it is not compatible with colabeling experiments involving immunofluorescence. To detect calcium deposition in fin fractures and skull injuries, live fish are incubated in calcein containing solution. Embedding, serial sectioning and staining of zebrafish.
Screening and mechanismanalysis in zebrafish for bone. A simple wholemount staining protocol for bone and or cartilage in adults and larvae clearing and staining for larval fish cartilage and bone chromosomes spreads. Any water stage is a good stopping point if you had to. Revisiting in vivo staining with alizarin red s a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration. I want to know if live cells are stained by calcein am, will. Lue1,2 the life scienceshoward hughes medical institute outreach program at harvard university supports.
Single staining methods using nucleic acid stains such as eb, pi, 7aad, dapi, sytox green, and sytox red, and enzymatic stains such as cfda and calcein am, were performed. Visualizing normal and defective bone development in zebrafish embryos using the fluorescent chromophore calcein. The present study is planned to investigate the effects of zinc chloride zncl2 on the bone embryonic development of danio rerio and confirm the use of zebrafish as a model organism to study the teratogenic potential of this pollutant. Characterization of zebrafish mutants with defects in bone. Dec 16, 2010 in this paper, we outline our protocol for embedding, serial sectioning, staining and visualizing zebrafish embryos embedded in jb4 plastic resina glycol methacrylatebased medium that results. Visualization of skeletons and intervertebral disks in. Alizarin 1,2dihydroxyanthraquinone, which emits a red signal under fluorescent green light, has been used for in vivo labelling for many decades.
We developed an assay calcein and alcian blue staining for screening zebrafish mutants with defects in bone development. This allows analysis of bone in live zebrafish with high sensitivity, though it also labels some cartilages at early stages. Pdf revisiting in vivo staining with alizarin red s a. Histological methods protocols zfin community wiki. Calcein is better retained in viable cells than are fluorescein, carboxyfluorescein and bcecf figure 15. Amongst dye reagents, including bcecf, am and carboxyfluorescein diacetate, calcein am is the most suitable indicator for staining viable cells due to its low cytotoxicity property. Visualization of skeletons and intervertebral disks in live fish larvae. Calcein violet am is optimized for use in flow cytometry. Ewes ovaries were harvested at the slaughterhouse, after dissection each ovarian specimen was divided into two groups. Agilents fish general purpose reagents are designed for use in fluorescence in situ hybridization fish protocols. Calcein staining trials using epifluorescence demonstrated the intense staining of coccolith calcite figures 1b,c that persisted for 24 h.
Calcein staining of red sea bream larvae successfully visualized development of craniofacial skeletons as well as urinary calculus. The data show a delay in hatching compared with the controls, malformations in the process of calcification and significant defects in growth. Fish were main tained as described in the zebrafish book 54. Examination of viability and quality of ovarian tissue after. In particular, we found three novel red fluorescent bonestaining dyes. Zebrafish as a model vertebrate for investigating chemical. For transmission electron microscopy of fish tissues, we generally prepare 4% glutaraldehyde in millonigs buffer. Calcein staining is a fluorescent dye used for detecting calcium as an.
Taking advantage of a highly conservative genomic, rapid development, large number of offspring, low cost and easy manipulation, zebrafish has been considered an excellent animal model for diseasebased drug screening. Calcein and alcian blue staining methods are quick and simple, highly sensitive, low toxic, and easy to detect. In addition, the fluorescence spectrum of calcein is similar to that obtained with fish tissue autofluorescence 29. Suitability of calcein as an in situ growth marker in. Feel free to add new protocols to the appropriate section or add comments to any existing protocol. A, b ventral view of the head skeleton of day7 a and 11 b embryos labeled with calcein. In addition, green fluorescent protein gfp reporter zebrafish was employed to trace lineage analysis of intervertebral disk cells in live fish larvae.
We demonstrated that ectopic expression of bmp2 in notochord cells inhibited the development of the axial skeleton. Calcein am is a widely used green fluorescent cell marker. The following is a staining protocol perfected by karen larison at zirc that we routinely use for zebrafish histopathology. Calcein staining is a fluorescent dye used for detecting calcium as an index of. Histological techniques are critical for observing tissue and cellular morphology. For zebrafish, only the use of calcein has been optimized for in vivo staining but most transgenic zebrafish lines use gfp as a reporter, which emits fluorescence within the same spectrum as calcein.
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