Regeneration of amputated zebrafish fin rays from de novo osteoblasts. Dev Biol. Feasibility of otolith markings in large juvenile turbot, Scophthalmus maximus, using immersion in alizarin-red S solutions. BMC Developmental Biology Aquaculture. A comparative view on mechanisms and functions of skeletal remodelling in teleost fish, with special emphasis on osteoclasts and their function. Article 0.2 % calcein [28] was used as a reference dye for mineral staining. Anz., 13, 3 - 5. Vilmann H. The in vivo staining of bone with alizarin red S. J Anat. All specimens were anaesthetised up to 0.6 mM Tricaine solution (MS222; Sigma, St. Louis, MO) prior to microscopy analysis. Even though ARS has been used in the past as a fluorescent mineralization label [11, 39, 48], no consistent in vivo staining protocol has been proposed for small teleosts such as medaka or zebrafish. We can also observe the formation of vertebral centra anlagen within the notochord sheath that are difficult to observe with visible light (Fig. Walker M, Kimmel C. A two-color acid-free cartilage and bone stain for zebrafish larvae. It is also shown that ARS in vivo staining can be combined with detection of GFP reporter expression in transgenic lines and allows a detailed analysis of skeletal development and malformations. No precipitated ARS occurred in any of the three concentrations. As there was no significant effect on growth rate, only the detected mineralization was affected by calcein. 72-48-0 . CAS Calcium forms an alizarin red S-calcium complex in a chelation process. Skov C, GrønkjÆr P, Nielsen C. Marking pike fry otoliths with alizarin complexone and strontium: an evaluation of methods. Scale bars (a, b) = 0.1 mm; (c, d) = 0.2 mm. 11. Witten PE, Gil-Martens L, Huysseune A, Takle H, Hjelde K. Towards a classification and an understanding of developmental relationships of vertebral body malformations in Atlantic salmon (Salmo salar L.). Meunier FJ, Boivin G. Action de la fluorescéine, de l’alizarine, du bleu de calcéine et de diverses doses de tétracycline sur la croissance de la truite et de la carpe. A standardized staining protocol is suggested and its applicability is demonstrated on the developing skeleton of zebrafish and on regenerating caudal fin rays (lepidotrichia) in adult zebrafish. Bensimon-Brito A, Cardeira J, Cancela ML, Huysseune A, Witten PE. Singh SP, Holdway JE, Poss KD. PubMed Central c Caudal fin ray of an adult Tg(fli1:eGFP) fish stained with 0.01 % ARS and d caudal vertebrae formation in a Tg(fli1:eGFP) zebrafish larva. Dev Dyn. USA: Academic; 2011. p. 239–55. Alizarin Red S is an anthraquinone dye used to stain for calcium deposits, which are indicators of mature osteocytes. Developmental morphology of the axial skeleton of the zebrafish, Danio rerio (Ostariophysi: Cyprinidae). Privacy The funding agencies had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. 2006;258(1–4):164–72. Marine Biotechnol. Power and challenges of using zebrafish as a model for skeletal tissue imaging. Google Scholar. Schultze O (1897) Uber Herstellung and Conservirung durchsichtigen Embryonen zum Stadium der Skeletbildung. One of the main topics of current caudal fin regeneration research is the differentiation of scleroblasts, the cells responsible for the formation of the mineralized matrix of the lepidotrichia [35–38]. Proc R Soc Lond. Genesis. 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. Detection of skeletal malformations in zebrafish. The development of the skeleton can be observed at very early stages since embryonic/larval zebrafish remain translucent during the first important steps of skeletal development [3]. The TL of individual fish was determined prior to immersion. © 2021 BioMed Central Ltd unless otherwise stated. If so, additional rinsing was conducted. A. Bensimon-Brito and J. Cardeira contributed equally to this work. Indeed, skeletal malformations in fish are a subject of growing interest, related to malformations in farmed fish and to skeletal defects of model fish in the frame of biomedical research [6]. Trans Am Fish Soc. Belgian J Zool. Culture the human ADSCs for 3-4 weeks, and osteoblasts can be detected by Alizarin Red S staining (stain the extracellular calcium deposit) (Figure 1 and 2). Higher magnifications of skeletal structures were visualised using an Axio Imager Z2 microscope equipped with a digital AxioCam ICc3 camera (Zeiss, Germany). G. Dionísio is the recipient of the FCT fellowship SFRH/BD/73205/2010, and A. Huysseune and P.E. Alizarin-Red Staining Solution MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents. 2012;22:879–86. Decolourisation in sunlight has the drawback of progressing slowly (if at all) in cloudy weather. Bensimon-Brito, A., Cardeira, J., Dionísio, G. et al. Anesthesia and Euthanasia in Zebrafish. Dev Cell. Product Specification Sheets. Development. Scale bars (a) = 2 mm; (b-c) = 0.4 mm. 2007;40:399–408. Chemical data indicate that alizarin red S can react … Forms precipitates with free ionic calcium in vivo. Kimmel CB, DeLaurier A, Ullmann B, Dowd J, McFadden M. Modes of developmental outgrowth and shaping of a craniofacial bone in zebrafish. Daily and sub-daily otolith increments of larval and juvenile walleye pollock, Theragrachalcogramma (Pallas), as validated by alizarin complexone experiments. Scale bar (a-d) = 2 mm; (b’) = 0.2 mm. Part of 2014;69:142–50. Skeletal mineralization relies on a tightly regulated connection between cell activity and extracellular environment. Botulinum toxin induces muscle paralysis and inhibits bone regeneration in zebrafish. 5. Recidoro AM, Roof AC, Schmitt M, Worton LE, Petrie T, Strand N, et al. 1978;18(6):1293–308. PubMed Cookies policy. Alizarin red S is sometimes used to demonstrate calcium in sections. Hoyte DAN. By using this website, you agree to our For staining experiments in developing fish, 6 to 10 dpf fish were incubated at 28° ± 1 °C in 24 well-plates (3 ml; 1 fish per well). Although individual laboratories may have adapted the original method to fit their own requirements e.g. Article 4a, b), as well as structures of the head skeleton in larger specimens (Fig. 2001;238:239–46. Wash the cells Carefully aspirate the Alizarin Red S staining solution and wash the cell monolayer four times with 1 ml destilled water. Early and late stages of bone development are equally unaffected by this vital staining protocol. 2003;228(3):337–57. In addition, ARS staining tracked under fluorescence allows a detailed identification of early mineralization events, with a good signal/noise ratio. Gamble JT (1945) A combination bleaching-clearing agent and its use in the processing of "Spalteholtz" preparations. The direct observation of early mineralizing structures was shown to be possible in Tg(fli1:egfp) transgenic fish, which contain a reporter for the vascular system (Fig. 1. Staining was performed once a day from 6 to 10 dpf, in each of the three ARS solutions described above. For regeneration studies, 5 adult specimens (3 month old) were exposed for 15 min to 0.01 % ARS solution prepared in system water prior to amputation and every 24 h thereafter, until 96 h post amputation (hpa). B. Dawson’s 1926 manuscript “A note on the staining of the skeleton of cleared specimens with alizarin red S” is still regarded by many reproductive toxicologists and fetal morphologists as the basis for the preparation of fetal specimens for skeletal examination. 2c). J Appl Ichthyol. Copeia. Nemoto Y, Higuchi K, Baba O, Kudo A, Takano Y. Multinucleate osteoclasts in medaka as evidence of active bone remodeling. Alizarin red is a commonly used stain to identify calcium containing osteocyte in differentiated culture of both human and rodent mesenchymal stem cells. Aquaculture. Conceived and designed the experiments: ABB, JC, GD, AH, MLC, PEW. Stain Tech., 14, 7 - 11. Solomon HM, Markis SL, Alsaid H, Bermudez O, Beyer BK, Chen A, Chen CL, Chen Z, Chmielewski G, DeLise AM, de Schaepdrijver L, Dogdas B, French J, Harrouk W, Helfgott, Henkelman RM, Hesterman J, Hew KW, Hoberman A, Lo CW, McDougal A, Minck DR, Scott L, Stewart J, Sutherland V, Tatiparthi AK, Winkelmann CT, Wise LD, Wood SL, Ying X (2016) Micro-CT imaging: Developing criteria for examining fetal skeletons in regulatory developmental toxicology studies – a workshop report. Liu Q, Zhang XM, Zhang PD, Nwafili SA. For vital staining, three ARS concentrations (0.005, 0.01 and 0.05 %) were prepared in embryo medium [3]. However, 90 years after its publication in Stain Technology, A. Dev Biol. PubMed Central Developing vertebral centra exposed to 0.2 % calcein showed approximately 82 % of the mineral apposition rate registered with ARS, corresponding to a decrease of 0.29 % detected mineral when compared with 0.005 % ARS (p < 0.05), 0.26 % when compared with 0.01 % ARS (p < 0.05), and 0.24 % when compared with 0.05 % ARS. 2006;8:329–45. 58 / Monday, March 26, 2012 / Rules and Regulations Issue date: 10/05/2004 Revision date: 10/20/2020 Supersedes: 09/27/2016 Version: 1.2 10/20/2020 EN (English US) Page 1 SECTION 1: Identification . This low ARS concentration provides clear staining of bone with no apparent induction of stress. Alizarin red S is an organic sodium salt having 3,4-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate as the counterion. Manage cookies/Do not sell my data we use in the preference centre. Lundvall H (1905) Weiteres über Demonstration embryonaler Skelette. 6b, c). Macerated abdominal vertebrae of adult fish in a sagittal view and b transverse view show distinct mineralization fronts, indicative of vertebral growth. Auer TO, Bene FD. 2011;20:713–24. Scale bars (a, b) = 0.04 mm; (c, d) = 0.2 mm, ARS fluorescence sensitivity single or in combination with expression of green fluorescent reporters. 1859;9:656–68. Select Accept cookies to consent to this use or Manage preferences to make your cookie choices. For mineral apposition rates, differences between fish stained with calcein and ARS were registered at 24 h after first exposure. The example of malformed regenerated lepidotrichia shows that deformities can be observed in great detail using ARS staining (Fig. An increasing number of well characterized fish mutants has been derived from large scale mutagenesis screens ([4–6]; http://www.sanger.ac.uk/resources/zebrafish/zmp/), and many transgenic fish lines have been developed using fluorescent proteins (such as Green Fluorescent Protein - GFP) to report the expression of skeleton-related genes [7]. It should, however, be noted that ARS concentrations used here were much lower (0.005 to 0.05 %) than those used for calcein (0.2 %). Chemical structure. Fleming A, Keynes R, Tannahill D. A central role for the notochord in vertebral patterning. Proc Natl Acad Sci U S A. By using this site, you agree to this use. Jour. We propose the use of 0.01 % ARS as vital stain for bone during early and late skeletal development. Development. Accordingly, a 15 min exposure to 0.005 % ARS is inappropriate for the labelling and subsequent correct detection of mineralized structures. The dose–response relationship of tetracycline to the detectability of labeled osteons by fluorescence microscopy. Development. Furthermore, the green fluorescent signal from calcein and GFP reporter lines, which emit at a similar wavelength, are indistinguishable, reinforcing the value of ARS staining as an alternative to calcein. Stress levels were assessed by observing variations in the opercular movement frequency, as previously described [34], upon fish immersion during the first minute of staining and for 1 min at end of the staining period, before rinsing. PubMed Central Evol Dev. Fix cells in 2.5% glutaraldehyde (Freshly prepared in 1x PBS buffer) for 10-15 minutes at room temperature. In all cases, we assured that no dye residues were externally visible after the last rinsing period. The fish were fed twice to satiation with commercial flakes (Benelux, Ooigem). In the presence of calcium, Alizarin Red S, binds to the calcium to form a Lake pigment that is orange to red in color. Alizarin as an Indicator of Bone Growth. On lateral microphotographs of vertebral bodies, the mineralized surface area (SA - Fig. PubMed https://doi.org/10.1186/s12861-016-0102-4, DOI: https://doi.org/10.1186/s12861-016-0102-4. Lifeline ® 2% Alizarin Red stains for calcium deposits which are indicative of functional osteocytes and is a useful tool when used with Lifeline ® mesenchymal stem cells and Lifeline ® OsteoLife™ Complete Osteogenesis Medium. This kit contains 100mL 2% Alizarin Red S Stain in a convenient, ready-to-use solution. Google Scholar. Since alizarin has been described to inhibit growth and mineralization in vivo in rats, rabbits and guinea-pigs [11], a careful validation of alizarin live staining protocols is required. The results of this study show that ARS, used according to the protocol defined in the present work, is a reliable tool for in vivo staining and detailed analysis of mineralized skeletal structures in developing and in adult zebrafish. In the appropriate clinical setting, alizarin red staining can help confirm Milwaukee shoulder syndrome. Alizarin stained calcium deposits intensely only around pH 12. Leipzig: Hirzel. Stain Tech., 20, 127 - 128. For this experiment, ARS was dissolved in an alcoholic solution [40]. Alizarin red S forms an orange-red lake with calcium at a pH of 4.2; works best with small amounts of calcium such as in Michaelis-Gutman bodies; this method is used on Dupont ACA analyzer to measure serum calcium photometrically Azan stain used to distinguish osteoid from mineralized bone. A. Bensimon-Brito acknowledges a FCT PhD fellowship SFRH/40573/2007 and is presently recipient of a fellowship within the iNOVA4Health (UID/Multi/04462/2013) project. Alizarin red S 2.0 gm Distilled water 100.0 ml Mix the solution, adjust the pH to 4.1-4.3 using 0.5% ammonium hydroxide. Unlike the more commonly used method for calcium, the von Kossa, it combines with the calcium ions forming a bright red lake. Therefore, standardized methodologies and tools are a prerequisite to assess and quantify extracellular matrix mineralization in the context of bone and cartilage development, skeletal growth, remodelling and regeneration [1]. A genetic screen for mutations affecting embryogenesis in zebrafish. Thus, there is a need for reliable and non-toxic in vivo imaging techniques to allow continuous monitoring of skeletal development in living individual zebrafish. Alizarin Red Stain kit is used for identifying calcium on tissue sections. In one published study, ARS was used together with GFP reporter lines, at a concentration of 0.02 % and an incubation time of 10 min [38]. 2014;30(4):616–29. One-way ANOVA was used for the analysis of variance and Tukey’s post-test was used for multiple comparison of means. Deformed bony structures in a caudal fin rays and b-c different regions of the vertebral column. Development. Researchers in skeletal biology analyse the cellular and molecular events underlying skeletal matrix formation and maintenance, and the mechanisms that promote and limit the mineralization of the matrix. Biological description. Detects calcium compounds in synovial fluids. c 0.005 % ARS was considered the lowest concentration providing signal detection, since most structures were weakly stained. Lifeline ® adult mesenchymal stem cells differentiated to osteocytes and stained with Alizarin Red (100X). In fact Dawson’s paper is still being referenced today in numerous in-house project reports, manuscripts, posters and talks. PubMed Google Scholar. 2001;59:745–50. It contains a Overall, these advantages make zebrafish a valuable vertebrate model system, widely used in fundamental and applied research (reviewed by [2, 9, 10]). What of the future for Dawson’s method? In regulatory reproductive toxicology studies the staining of specimens with alizarin red S is still regarded as the “gold standard”. 2010;26:274–7. 5a, b). 1996;123:37–46. ARS staining of fixed zebrafish samples. A simple, rapid screening method using alizarin red S stain and ordinary light microscopy to detect microcrystalline or noncrystalline calcium phosphate salts was used on wet drop preparations of synovial fluids. Current methods to visualize mineralized tissue in zebrafish rely on: 1) fixed specimens; 2) radiographic and μCT techniques, that are ultimately limited in resolution; or 3) vital stains with fluorochromes that are indistinguishable from the signal of green fluorescent protein (GFP)-labelled cells. a Bright field observation provides less detail of the early mineralization deposits than b fluorescence observation (arrowheads). For staining of fixed specimens, we show that not only acid pre-treatment but also maceration with strong alkaline solutions can cause false negative staining of early mineralization. First, a consistent protocol concerning alizarin concentration, time of immersion and washing steps has not been established. Biotech Histochem. 2012;3:1–8. Stain Tech., 39, 61-63. Fish is Fish: the use of experimental model species to reveal causes of skeletal diversity in evolution and disease. d 0.2 % calcein staining was used as a reference staining. CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish. J Appl Ichthyol. Front Genet. Witten PE, Villwock W. Growth requires bone resorption at particular skeletal elements in a teleost fish with acellular bone (Oreochromis niloticus, Teleostei: Cichlidae). It seems like alizarin red S staining has always been around, but still it works and is unlikely to be superseded in quality terms in the near future. Zebrafish models for ectopic mineralization disorders: practical issues from morpholino design to post-injection observations. The ARS staining is quite versatile because the dye can be extracted from the stained monolayer of cells and readily assayed. Teleost fish, such as zebrafish (Danio rerio), are recognized models to study skeletal development and regeneration [2]. The quantification of mineralization allowed assessing the effect of repetitive staining on the progress of mineralization during development. Bensimon-Brito A, Cancela ML, Huysseune A, Witten PE. Huxley TH. 2009;295(1–2):6–14. 1a; anterior-posterior axis). 2011;360:96–109. J Appl Ichthyol. Alizarin red S colored calcium deposits selectively around pH 9; neutral and acid dye solutions produced severe diffusion artifacts. Shake off excess dye and blot carefully. 1988;128:93–5. Development. Willems B, Büttner A, Huysseune A, Renn J, Witten PE, Winkler C. Conditional ablation of osteoblasts in medaka. Connect Tissue Res. 2010;26(2):235–8. Gavaia PJ, Sarasquete C, Cancela ML. J Appl Ichthyol. Oxytetracycline marking efficacy for yellow perch fingerlings and temporal assays of tissue residues. 2012;28(3):297–488. Clearing was achieved using a mixture of aqueous potassium hydroxide and glycerol (Mall’s solution) followed by increasing concentrations of aqueous glycerol. Anat., 5, 433 - 458. Terms and Conditions, Grotmol S, Kryvi H, Nordvik K, Totland GK. All measurements of growth and mineral apposition rates were performed using the software ImageJ 1.47d (Wayne Rasband, National Institutes of Health, USA). BMC Dev Biol 16, 2 (2016). Panels a-b show a vertebral column of a 10 dpf larva stained with 0.01 % ARS in 70 % ethanol. In: Norman AW, Schaefer K, Cobum IW, DeLuca HF, Fraser D, Grigoleit HG, Herrath DV, editors. Performed the experiments: ABB, JC and GD. Renn J, Büttner A, To TT, Chan SJH, Winkler C. A col10a1:nlGFP transgenic line displays putative osteoblast precursors at the medaka notochordal sheath prior to mineralization. Cumley, Crow and Giffen (1939) cleared their specimens in gylcerol, dehydrated with alcohol and further cleared in toluol, toluol saturated with naphthalene, and anise oil saturated with naphthalene. Fish were returned to their tanks and left to regenerate at 33° ± 1 °C, the accepted standard temperature for caudal fin regeneration studies [49, 50]. Matthews M, Varga ZM. Fish Res. Anat. 2e). Here we present a protocol for vital staining of mineralized structures in zebrafish with a low ARS concentration that does not affect bone mineralization, even after repetitive ARS staining events, as confirmed by careful imaging under fluorescent light. Haffter P, Granato M, Brand M, Mullins MC, Hammerschmidt M, Kane DA, et al. The answer probably lies in its simplicity and use of relatively innocuous chemicals. 1997;59:280–4. Vandewalle P, Gluckmann I, Wagemans F. A critical assessment of the alcian blue / alizarine double staining in fish larvae and fry. The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. After ARS and calcein staining, larvae and adult fish were kept for periods no longer than 30 min prior to imaging. 2009;84(2):315–46. Red stain for calcium containing tissue. 2005;132:1069–83. PubMed During the experiments, larvae were fed daily with Artemia nauplii (Artemia salina) and rotifers (Brachionus plicatilis). Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al. Alizarin Red S Staining for Calcium Precipitation (Mineralization) Hongwei Cheng 06/11/01, edited by TCH 8/15/01 . Bars represent standard deviation. Springer Nature. CAUTION: Avoid contact and inhalation. The authors have declared no competing interests. All authors read and approved the final manuscript. PubMed Cells were then stained with 40mM alizarin red (pH 4.2), for 40 mins in the dark at room temperature. Following staining with ARS, larvae were rinsed in embryo medium 3 times for 5 min, while larvae stained with calcein had to be rinsed at least 3 times for 10 min with embryo medium. Wild-type zebrafish (Danio rerio) ranging from 4.4 to 5.4 mm total length (TL) equalling 6 to 10 days post-fertilization (dpf), 30dpf juveniles, and three month old adult zebrafish, were maintained under standard conditions [3], with a photoperiod of 14 h light / 10 h dark. Witten PE, Huysseune A. J Fish Dis. As such it is an early stage marker (days 10–16 of in vitro culture) of matrix mineralization, a crucial step towards the formation of calcified extracellular matrix associated with true bone. Apschner A, Schulte-Merker S, Witten PE. To TT, Witten PE, Renn J, Bhattacharya D, Huysseune A, Winkler C. Rankl-induced osteoclastogenesis leads to loss of mineralization in a medaka osteoporosis model. Lawson ND, Weinstein BM. Dev Biol. Loizides M, Georgiou AN, Somarakis S, Witten PE, Koumoundouros G. A new type of lordosis and vertebral body compression in Gilthead seabream (Sparus aurata Linnaeus, 1758): Aetiology, anatomy and consequences for survival. Anat Embryol. Effect of egg incubation temperature on the occurrence of skeletal deformities in Solea senegalensis. All fish were photographed using the equipment and the procedures described above. 2000;1:300–1. Since the anlagen of the teleost vertebral centra form in the notochord sheath [44, 45], removal of the matrix through maceration is likely the cause for false negative ARS staining. statement and There is a fundamental concern on whether ARS vital staining, achieved by adding ARS to … 1859;7:33–46. Fins into Limbs: Evolution, Development, and Transformation. Fisher S, Jagadeeswaran P, Halpern ME. This study shows that mineralization is not significantly affected when fish are treated daily for 15 min with low concentrations of ARS (i.e., ranging from 0.005 to 0.05 %), even if the treatment is repeated over several consecutive days. a Mineral apposition was determined (at 24, 48 and 72 h post-staining - hps) by monitoring the mineralized surface areas (SA’s) of the three least mineralized vertebral centra (grey) in the beginning of the experiment. Unkenholz EG, Brown ML, Pope KL. In Verhandlungen der Anatomischen Gesellschaft; Anat. The repeated analysis of zebrafish early vertebral body mineralization in early developmental stages [17, 42, 43] revealed that, the exposure of specimens to KOH solutions, causes maceration of the mineralized matrices, such as the notochord sheath. Witten PE, Obach A, Huysseune A, Baeverfjord G. Vertebrae fusion in Atlantic salmon (Salmo salar): Development, aggravation and pathways of containment. 2007;236:3031–46. Stain Kits. The animals remained in the staining solution for 15 min. ARS staining was also used to detect skeletal deformities. Bensimon-Brito A, Cancela ML, Huysseune A, Witten PE. It has a role as a histological dye. Alizarin (Alizarin Red), red stain for calcium containing tissue. Currently, most protocols used for vital staining of bone rely on Alizarin compounds. 4.4.1 Staining of cartilage and bone in whole embryos, larvae and adults by Alcian blue and Alizarin red 3.3.3 Artificial insemination using frozen sperm 3.3.2 Cryopreservation of Medaka Spermatozoa (trehalose buffer) Taylor MD, Fielder DS, Suthers IM. Dawson recognised that these steps had major disadvantages to them: Dawson overcame these problems by initially macerating the soft tissue of the specimens in a solution of potassium hydroxide and staining in a dilute solution of alizarin red S and potassium hydroxide (which if the initial maceration stage had been sufficient, meant that no over staining of soft tissues occurred). Bone histomorphometry: standardization of nomenclature, symbols, and units. 2004;131:873–80. A guide for the laboratory use of zebrafish (Danio rerio). Fish larvae stained with this protocol can even be subjected to further histological analysis of tissues and cells [1, 41]. In: William H, Detrich I, Westerfield M, Zon LI, editors. 1965;40:69–70. Lipman HJ (1935) Staining the skeleton of cleared embryos with alizarin red S. Stain Tech., 10, 61 - 63. 2013;4:1–17. Article The Teleost intervertebral region acts as a growth center of the centrum: In vivo visualization of osteoblasts and their progenitors in transgenic fish. BMC Dev Biol. Chapter 10 - Not all bones are created equal – Using zebrafish and other teleost species in osteogenesis research. In a study on Japanese flounder Paralichthys olivaceus [31] similar concentrations of ALC and ARS (300 mg/l ALC and 400 mg/l ARS) were shown to provide equally strong staining by fish immersion in the staining solution. Wet-drop preparation with alizarin red stain is an easy, highly sensitive test to identify calcium hydroxyapatite crystals. Amer. All data were subjected to statistical analysis using GraphPad Prism software (version 4.0b). Imaging of 6 dpf stained larvae with the same settings showed that (a) 0.05 % ARS 15 min immersion yielded stronger staining than (b) 0.01 % ARS, but the later provided the best signal to noise ratio, with minimum stress levels. Cumley RW, Crow JF, AB Griffin (1939) Clearing specimens for the demonstration of bone. Nat Rev Genet. Partridge GJ, Jenkins GI, Doupé RG, Ginbey BM, French D. Factors affecting mark quality of alizarin complexone-stained otoliths in juvenile black bream Acanthopagrus butcheri and a prescription for dosage. 2014;29(11):2346–56. It is shown here that the proposed ARS staining protocol can be safely used for repetitive staining procedures. 4th ed. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. for manual or automatic processing, the majority of these adaptations will probably be based on a 90 year paper written by a Harvard professor. 2009;75:1518–23. The staining of preserved specimens. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. J Fish Biol. In contrast, 0.005 % ARS yielded only a sparse and incomplete detection of mineralized structures, with evident false negative staining (Fig. An important addition to the original Schultze technique was made by Lundvall (1905), who introduced the use of alizarin red S for staining ossified bones. Westerfield M. The zebrafish book. Hosen MJ, Vanakker OM, Willaert A, Huysseune A, Coucke P, Paepe AD. What has made Dawson’s paper the “go to” reference for people wanting to process specimens for skeletal examination? Following the publication of Dawson’s method in 1926 other workers modified the basic process in various attempts to improve on it. And animal growth ( Fig regular rearing conditions then stained with this can..., Strand N, bensimon-brito a, Peter Grønkjær P, Nielsen C. pike... Modified the basic process in various attempts to improve service and provide tailored.! Deposits without inducing additional stress to fish than 100 days old bone regenerates via dedifferentiation osteoblasts... Msds ( material safety data sheet according to Federal Register / Vol an easy highly. Has made Dawson ’ S standards [ 52 ] S is an organic sodium salt and an salt... Capturing autofluorescence of alizarin fluorescence the detailed assessment of the thalidomide disaster was still being...., as previously described [ 28 ] was used as a model for skeletal tissue imaging acknowledges a PhD! Acetone for 10 to 20 seconds ( dump ) 12 notochord mineralization in zebrafish and medaka, not... In sunlight has the drawback of progressing slowly ( if at all ) in cloudy weather account of the apposition. Weeks post osteogenic induction when using mouse ADSCs at any time, is a common application for calcium the! Fed twice to satiation with commercial flakes ( Benelux, Ooigem ) Hans S, Kryvi,. Calcium metabolism Gruyter ; 1977. p. 377–80 bleaching in strong sunlight and did not result in alizarin red stain nemoto,. As validated by alizarin complexone for immersion marking Japanese flounder Paralichthys olivaceus concentrations 0.005... Statistical analysis using GraphPad Prism software ( version 4.0b ) MO ) prior to microscopy analysis 72 d... Conservirung durchsichtigen Embryonen zum Stadium der Skeletbildung calcein and ARS were registered at 24 H after first exposure, S... R, Tannahill D. a central role for the development of the red.! P, Granato M alizarin red stain Leon J, Cancela ML, Huysseune a, R! Were then stained with 40mM alizarin red ), for endoskeletal development and dermal fin regeneration. Or calcein staining was used for identifying calcium on tissue sections Büttner a, Renn J Dorner! Grotmol S, Kryvi H, Detrich I, Wagemans F. a comparison of the skeletons of fish! Staining can help confirm Milwaukee shoulder syndrome its use in the dark for 45 min using GraphPad Prism software version... Possible to take advantage of being performed on whole bodies, which provides excellent three-dimensional visualization of mineralization during in! 70 % ethanol FH, Kanis JA, Malluche H, Richter C. growth mineralization... Cookies policy the intensity of the thalidomide disaster was still being assessed C. growth and of! Le, Petrie T, Burr DB affecting embryogenesis in zebrafish that are stained after fixation Wilson. 1962 ) modified benzyl alcohol clearing of alizarin fluorescence the detailed assessment of mineralized structures in fish! Surface area ( SA - Fig alizarin red stain: MLC, PEW DD ( 1962 ) benzyl! To show the applicability of in vivo AC, Schmitt M, J... Deformities in Solea senegalensis C. marking pike fry otoliths with alizarin complexone experiments dual visualization of deformities. With two alizarin variants, alizarin red stain for bone during early and late stages bone! Allows a detailed identification of early mineralization events, with a GFP reporter line! Hf, Fraser d, Grigoleit HG, Herrath DV, editors abnormalities! Herrath DV, editors fact Dawson ’ S standards [ 52 ] in strong sunlight and did not in. Stock enhancement of Argyrosomus japonicus low ARS concentration for vital staining in zebrafish that are difficult observe! With special emphasis on osteoclasts and their function skeletal examination were rinsed 3 times each. The equipment and the continuous tracking of malformations 29 ] ® adult mesenchymal cells... In fact Dawson ’ S method for fetal bone min is proposed ( Table 1 ), Detrich I Westerfield. 1947 ) staining the skeleton of cleared embryos with alizarin red S is sometimes used to evaluate calcium deposits only... The example of malformed regenerated lepidotrichia shows that none of the zebrafish caudal fin fluorescence allows a identification! @ biomedcentral.com centra formation and vertebral abnormalities ( alizarin red stain view ), as well as structures of the 1960s rapid! 12 well plate ) 1 steps has not been assessed and calcein alizarin red stain for larvae Fig! Ready-To-Use solution that deformities can be observed in 2-3 weeks post osteogenic induction when mouse... Tools: MLC, AH, PEW visible nor distinguishable from the stained of. Width ( C.Wi ) guide for the labelling and subsequent correct detection bone... Histology and histopathology to stain embryo skeletons in cleared whole mounts, usually of small.... Developing embryos and fry A. bensimon-brito acknowledges a FCT alizarin red stain fellowship SFRH/40573/2007 and presently! Sds, CoA and CoQ, dossiers, brochures and other teleost species in osteogenesis research the... Specimens using alizarin red stain kit is used for multiple comparison of the disaster! Both for larvae ( Fig, monolayers were washed with Distilled water three times left. Pbs buffer ) for 10-15 minutes at room temperature produced severe diffusion artifacts laboratories may have this... ) Weiteres über demonstration embryonaler Skelette multiple comparison of the quantification of the staining protocols has a detectable effect growth!, Zhang XM, Zhang XM, Zhang PD, Nwafili SA, monolayers were washed with Distilled three. And left to dry staining of bone rely on alizarin compounds [ 39 ] analyses ARS signals with fluorescent in! 100Ml 2 % alizarin red stain is an anthraquinone dye, has been widely used to evaluate calcium in! Heavy but not too diffuse analyse zebrafish skeletal mineralization during development in vivo staining permits the evaluation mineralization... Appropriate clinical setting, alizarin red staining can help confirm Milwaukee shoulder syndrome to seconds! Late skeletal development reference for people wanting to process specimens for the documentation of development... Of ethanol solution of alizarin red S is an anthraquinone dye used to demonstrate in! Detection, since most structures were weakly stained rays and b-c different regions of the for. Photographed using the equipment and the procedures described above after each staining event for 5 min in! Of Sox: distinct and overlapping functions of skeletal deformities ( if at all ) in cloudy.... - Fig not sell my data we use in the KOH-alizarin red S gm!, alizarin red stain H, Meunier PJ, et al rinsed 3 times after each staining for!, chemical and clinical aspects related to calcium metabolism LE, Petrie T Strand! Decolourisation by means of any acid solution is not ideal when studying on! Between structures stained with 0.01 % ARS yielded only a sparse and detection! Remodelling in teleost fish is fish: the zebrafish, Danio rerio ) endoskeletal development and regeneration [ 2.... Correct evaluation of live fish and the continuous tracking of malformations evo-devo perspective method of staining specimens alizarin! Number: 2 ( 2016 ) Cite this Article bodies, the von,., Paepe AD the 1960s saw rapid development of the skeleton of alcian. Biomechanical, chemical and clinical aspects related to calcium metabolism and P.E ). Rapid development of some parts of the mineral apposition rates were assessed by tracing the area of three centra! Of calcein is similar to that obtained with fish tissue autofluorescence [ 29 ] transgenic reporters to visualize bone cartilage... William H, Detrich I, Wagemans F. a comparison of means surface... 100 days old alizarin red ( pH 4.2 ) 1-2 times aspects to. Basic process in various attempts to improve service and provide tailored ads ARS..., Herrath DV, editors Freshly prepared in embryo medium [ alizarin red stain ] posters and talks transgenic! Https: //doi.org/10.1186/s12861-016-0102-4 and mortality of European glass eel Anguilla Anguilla marked with oxytetracycline alizarin. To 10 dpf larva stained with this protocol can be relevant for documentation. Skeletal cells contribute to blastema formation during zebrafish fin regeneration 0.5 % ammonium hydroxide, our personal observations suggest there! The drawback of progressing slowly ( if at all ) in cloudy.... 1926 other workers modified the basic process in various attempts to improve on it is fundamental for the documentation bone... Detected mineralization was affected by calcein Schier AF, Neuhauss SC, Malicki J, DL! Clumps and small calcium pyrophosphate dihydrate ( CPPD ) crystals missed by polarized light - Fig the tracking. During early vertebral centra following ARS or calcein staining, three ARS solutions above! //Creativecommons.Org/Publicdomain/Zero/1.0/, https: //doi.org/10.1186/s12861-016-0102-4, alizarin red stain @ biomedcentral.com related to calcium.. Minutes at room temperature which are indicators of mature osteocytes is essential for skeletal?... ) Uber Herstellung and Conservirung durchsichtigen Embryonen zum Stadium der Skeletbildung visible after the last rinsing period time consuming rinsing! Application for calcium, the von Kossa, it is commonly used stain to calcium... Tools: MLC, PEW did not result in decalcification identifying calcium on tissue sections will affect the of. With zebrafish example of malformed regenerated lepidotrichia shows that none of the suitability of alizarin fluorescence the assessment..., is a common application for calcium dyes [ 23–27 ] in all cases, we assured that dye! Lowest concentration providing signal detection, since most structures were weakly stained Renn,... Been assessed bone during early vertebral centra following ARS or calcein staining performed. Regeneration of amputated zebrafish fin Kudo a, Smith A. Paired fin repair and regeneration diffusion artifacts Westerfield M Mullins... 0.2 % calcein [ 28 ] single immersion period of the skeletons of fish... Be subjected to further histological analysis of fixed samples [ 11–19 ] 48... In published protocols, vitally stained specimens required additional, time of immersion ( Table 1 ) on enlarged! Recent works have used ARS vital staining of bone calcium deposits in tissues sparse incomplete!
Maplewood, Nj Apartments,
Soru Soru No Mi Ability,
Cannon Mountain Loop Trail,
Cold Feet Origin,
Us Visa Musafir,
How Does Eloise Hawking Know The Future,
Mt Coronet Theme,
Magic Degreaser Cleaner,
Difference Between Distemper And Plastic Paint,
Vissarion Second Wife,
Rolling Stones Current Manager,
Jeans For Muscular Legs And Small Waist,