01, · Calcein staining. Calcein solution (0.2) was prepared by dissolving 2 g of calcein powder (TCI C0004) in 1 litre of deionized water (pH 7.4). Zebrafish larvae were treated wi 0.04 calcein in E3 medium for min, washed in E3 medium ree times, and rinsed in Cited by: 12. Apr 13, 20 · Calcein staining of red sea bream larvae successfully visualized development of craniofacial skeletons as well as urinary calculus. Histochemical detection of alkaline phosphatase (ALP) activity revealed at abnormal segmentation of notochord induced by RA during vertebral development in zebrafish. Immunohistochemistry clearly revealed at GFP‐positive cells in Cited by: 6. 01, · Staining wi Calcein-AM, a cell-permeant dye which labels live cells wi green fluorescence (Feller et al., 1995), showed at all adherent cells were viable. e strong fluorescence signal of Calcein-AM allowed for e observation of differentiated cells, i.e. polarized neuron-like cells, already at 1 dap (Fig. 1 D).Cited by: 7. 11, · Zebrafish larvae (5–13 dpf) were stained wi calcein for 15 min at room temperature. After removing calcein solutions, e fish were washed 2–3 times wi system water ( min per wash), anes etized wi 0.1 e yl 3-aminobenzoate me anesulfonate salt, viewed and imaged using a dissecting fluorescence microscope.Cited by: . Vital staining wi Calcein green and Alizarin red to label mineralized matrix. Nine wild type zebrafish (21 dpf) wi SL between 4.5 mm and 9 mm were used for live staining as described by. 0.003 Alizarin red and 0.0025 Calcein green stains were applied sequentially for overnight incubations using water from e fish facility. ree different conditions were tested. In situ hybridisation detects OCRL expression in e glomeruli of zebrafish and adult human kidney tissue. a Top panels show OCRL expression in a whole-mount zebrafish embryo (left panel, lateral view, anterior to e left) and in transverse section rough e glomerulus (right panel, highlighted wi white dotted outline) at 5 days post fertilisation (dpf). b In situ hybridisation of normal. Calcein AM is a cell-permeant dye at can be used to determine cell viability in most eukaryotic cells. In live cells e nonfluorescent calcein AM is converted to a green-fluorescent calcein after acetoxyme yl ester hydrolysis by intracellular esterases. is dye is also available as 1 mg of e solid (C-1430) and resuspended in DMSO (C-3099). We are staining our endo elial cells wi e live cell staining calcein. Due to a time-consuming experimental set-up, it would be very helpful if e cells could be fixated after calcein-staining. We demonstrated at ectopic expression of BMP2 in notochord cells inhibited e development of e axial skeleton. Toge er, ese results clearly demonstrated e sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants at have bone structure defects. 19, · Here, a daily, single immersion period of 15 min is proposed bo for larvae (Fig. 1) and adult zebrafish. Calcein, ano er standard reagent for in vivo skeletal staining of zebrafish, was used as a control staining, following an established calcein staining protocol. It should, however, be noted at ARS concentrations used here were much lower (0.005 to 0.05 ) an ose used for calcein . 14, · is is where zebrafish researchers can share experimental protocols and tips wi e rest of e research community. Protocols are organized into sections corresponding to e chapters of e Zebrafish Book, 5 edition (4 edition on-line). Feel free to add new protocols to e appropriate section or add comments to any existing protocol. For zebrafish, only e use of calcein has been optimized for in vivo staining  but most transgenic zebrafish lines use GFP as a reporter , which emits fluorescence wi in e same spectrum as calcein. In addition, e fluorescence spectrum of calcein is similar to at obtained wi fish tissue autofluorescence . us, alternatives to calcein. Apr 06, · e following is a staining protocol perfected by Karen Larison at ZIRC at we routinely use for zebrafish histopa ology. Any water stage is a good stopping point if you had to do some ing part way rough e procedure. Keep e slides wet roughout e procedure. Calcein Staining. Calcein (C 30 H 26 N 2 O 13) is a fluorescent chromophore at specifically binds to calcium. As e skeletal system contains calcified structures, calcein has been used to label bone structures and to study bone grow (Ducy et al., 2000). To stain in situ, we recommend e following protocol:. Remove media and wash e cells once wi serum-free buffer to remove any remaining media. 2. Add dye diluted in serum-free buffer to e culture.. We recommend staining at a 1- μM concentration of BD Pharmingen Calcein AM dye for live/dead cell discrimination. 08, · e calcein staining was excited at 488 nm and e emission was collected at 492/577 nm. Z-stacks were acquired at 1.5 μm increments, every 1 min. Pictures were processed off-line using ImageJ (NIH) and Avizo (FEI). 4 zebrafish larvae were observed for each condition and time. Figure. Evaluation of mineralization in alendronate-treated zebrafish. (A) e gross morphology of zebrafish aged at 7 dpf which have been treated wi different concentrations of alendronate (, 20, and 30 µM, left bright-filed panel) from 3 dpf onds. Calcein staining on control. Calcein (Sigma-Aldrich, C0875-56) staining, used to label e regenerated bony-rays, was performed as previously described (Du et al., 2001). Fins were imaged using a Zeiss Lu V-12 fluorescence stereoscope equipped wi a Zeiss digital camera using a 0.8× . 16, 2006 · e labile iron pool is a putative cytosolic compartment of loosely bound, redox-active, chelator-accessible iron. Iron contained wi in is pool is ought to influence e activity of iron regulatory proteins (IRPs), which bind to iron response elements (IRE) during low iron conditions. is association blocks e translation of ferritin mRNA, and stabilizes transferrin receptor mRNA. Hoechst staining of isolated ctsk + cells in adult zebrafish skeletal tissue demonstrated e recovery of a wide diversity of single and multinucleated cells (Fig. S4E,F). e presence and activity of bo populations of cells in tissues in situ can be detected rough analysis of e activity of tartrate-resistant acid phosphatase (TRAP) using. Over e past ades, studies using zebrafish have significantly advanced our understanding of e cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos at allow comprehensive imaging of embryogenesis combined wi powerful genetic approaches. However, ford genetic screens in zebrafish have generated unanticipated findings . Calcein [C001, Dojindo, Japan. stock solution (1 mg/ml) was prepared by dilution from stock (50 mg/ml) in 1 M KOH] was incubated at 1 μg/ml for Ca 2+ imaging during culture wi normal or osteogenic media. For mineral staining, cells were fixed wi cold 0 e anol for 5 min and stained wi 1.0 Alizarin Red S solution (pH adjusted to 6.4. 18, · FIGURE. Calcein staining of calcium carbonate coccoli s using epifluorescence. (A) e structure of calcein (Bis[N,N-bis(carboxyme yl)aminome yl]fluorescein) wi four carboxyl groups at coordinate and bind wi Ca 2+ ions, ereby altering fluorescence properties. (B) Scyphosphaera apsteinii, from left to right. SEM, DIC micrograph and corresponding calcein epifluorescence (FITC. Calcein staining of young zebrafish. Calcein is a fluorescent chromophore which binds specifically to calcified skeletal structures and allows e screening of skeletal mutants. Micr scan of an adult zebrafish (Bruker, 1172). We use micr analysis to determine Bone Mineral Density in zebrafish. Alizerin red staining of a 22 days old zebrafish. 1 Article 2 Live Fluorescent Staining Platform for Drug- 3 Screening and Mechanism-Analysis in Zebrafish for 4 Bone Mineralization 5 g-Ren Chen 1, Yu-Heng Lai2, Jhih-Jie Tsai 3 and Chung-Der Hsiao 3,4,5 * 6 1 Department of Biological Science & Technology College of Medicine, I-Shou University, Kaohsiung, 7 Taiwan. [email protected] 8 2 Department of Chemistry, Chinese Culture University. A Simple Whole-Mount Staining Protocol For Bone And Or Cartilage In Adults And Larvae. Clearing And Staining For Larval Fish Cartilage And Bone. Chromosomes Spreads. Gentle Fixation By Freeze Substitution Gives Excellent Histological Results Wi Zebrafish Embryos. Zebrafish Monoclonal Antibodies. Zebrafish TEM. 3 Micron Cryosections. In vivo bone staining was modified from e technique described for zebrafish by Du et al.: mg calcein powder (Sigma Chemical Co.) was dissolved directly into 0 ml saltwater to make a 0.01 solution, en passed rough a 5 μm filter to remove insoluble particles. live larvae were incubated in e solution (-20 min), followed by ree. Zebrafish have an unlimited capacity to regenerate bone after fin amputation. In is process, mature osteoblasts dedifferentiate to osteogenic precursor cells and us represent an important source of newly forming bone. By contrast, differentiated osteoblasts do not appear to contribute to repair of bone injuries in mammals. ra er, osteoblasts form anew from mesenchymal stem cells. Calcein information Calcein staining of fish to be released into e wild is not yet approved for general use. It is being studied as an Investigational New Animal Drug (INAD), collecting e data at will be used to support a new animal drug application (NADA). e zebrafish is an ideal model at could link preclinical toxicity screening wi e drug development pipeline. 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 disease-based drug screening. Fig. 2. External features of developing larvae and internal ossification of e same individuals revealed by calcein staining. Larvae are not to scale. - Normal table of postembryonic zebrafish development: staging by externally visible anatomy of e living fish.. Co-staining wi Annexin V or 7-AAD is recommended to allow e greatest resolution between live and dead/apoptotic cells. If single-color analysis is desired, improved resolution between live and dead/apoptotic cells be obtained by using ei er Calcein Blue AM (cat. 65-0855) or Calcein Violet 450 AM (cat. 65-0854). Molecular weight: 994.86. 09, · We compared calcein staining of e skeletons of e wild type and nacre mutants, which are transparent zebrafish, wi micro-CT for e first . e chaperone activity o PBA ameliorates e skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta Hum Mol Genet. 1.26(15):2897-2911. doi: . 93/hmg/ddx171. staining was also evident around e notch tip (Fig. 2b). No differences were observed between BaSO4 (Fig. 2) and calcein (not shown) stained specimens, and no cracks or bright lines were observed in unloaded control specimens (Fig. 2a). Cracks pri ily propagated along e same pa as e bright lines of un-notched specimens, perpendicularly. Currently, drug screening relies on cell-based experiments or on animal models to confirm biological effects. e mammalian system is considered too time-consuming, expensive and complex to perform high- roughput drug screening. ere is a gap between in vitro cell-based models and e in vivo mammalian models. e zebrafish is an ideal model at could link preclinical toxicity screening. 1X Calcein AM DW Buffer - Dilute X Calcein AM DW Buffer 1: in deionized sterile water to make 1X Calcein AM DW Buffer. For each 96-well plate, use 5.0 mL of X Calcein AM DW buffer and 45 mL of deionized sterile water. Calcein AM Stock Solution- e molecular weight of Calcein . staining dyes: purpurin, lucidin and 3-hydroxy-morindone. purpurin can effectively label bones in bo larval and adult zebrafish, as well as in postnatal mice, wi out significantly affecting bone mass and density. Moreover, two structurally similar chemo erapeutic compounds, doxorubicin and epirubicin, were observed to have. Cellular Viability Calcein / Propidium Iodide We’ve performed is assay on e following cell types: • Dissociated Pri y Cortical Neurons Plated at 0.2 - 1.0x 6 cells/ml (0 μl/well of 96 well plate. 1 ml/well of a 24 well plate. 4 ml/well of 6 well plate). Because Calcein AM is photostable, shows low cytotoxicity, does not affect cellular function, and requires cellular esterases for conversion to green fluorescing Calcein, it is a popular stain for e examination of cell vitality and viability (1,2,3). Braut-Boucher, F. et al. Journal of . e emerging fungal pa ogen Candida auris has produced numerous outbreaks of invasive disease in hospitals worldwide. Why is species causes deadly disease is unknown. Our findings reveal a failure of neutrophils to kill C. auris compared to e most commonly encountered Candida species, C. albicans. While neutrophils produce neutrophil extracellular traps (NETs) upon encounter wi C. Calcein staining and calcium and phosphorus determination revealed at BA ameliorates mineralization of DEX-induced osteoporosis zebrafish larvae. BA also regulates e expression of RANKL and OPG and hampers e changes in gene expression related to bone formation and resorption under e induction of DEX in zebrafish. Washington University in St. Louis is now home to one of e largest zebrafish facilities in e world. Wi its robotic feeding and cleaning systems, it is e world’s most modern, says Lilianna Solnica-Krezel, PhD, professor and head of e Department of Developmental Biology at Washington University School of Medicine in St. Louis. Calcein is a calcium-dependent fluorescent molecule. It is mainly required to study bone metabolism under in vivo conditions and helps in e staining of pit area under in vitro conditions. Calcein is used for e fluorometric determination of calcium and EDTA (e ylenediaminetetraacet ic acid) titration of calcium. 1 E2/25/11v1 I. Introduction Trevigen’s Calcein AM Cell Viability Kit provides a simple, rapid and accurate me od to measure cell viability and/or cytotoxicity. Calcein AM (structure A) is a non-fluorescent, hydrophilic compound at easily permeates intact, live cells. By Jeremie Silvent (4672372), Anat Akiva (4672375), Vlad Brumfeld (1688731), Natalie Reznikov (4672369), Katya Rechav (1450945), Karina Yaniv (3475259), Lia . Larval zebrafish wi calcein stain. Larval zebrafish under microscopy. Larval shortnose sturgeon. Micromanipulation of zebrafish embryos, Zen at work and Optokinesis (OKR) system. Zebrafish rearing system. Winter flounder rearing. Recirculation system during experimentation. Shortnose sturgeon. Lab crew over e years.