a Bright field observation provides less detail of the early mineralization deposits than b fluorescence observation (arrowheads). Kölliker A. Taylor MD, Fielder DS, Suthers IM. J Bone Miner Res. Alizarin as an Indicator of Bone Growth. Performed the experiments: ABB, JC and GD. TL was measured every day and the growth rate was calculated based on TL measured at each time point divided by TL at the beginning of the experiment. To eliminate the background staining, calcein stained specimens required additional, time consuming, rinsing steps. ARS staining was also used to detect skeletal deformities. 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]. Unlike the more commonly used method for calcium, the von Kossa, it combines with the calcium ions forming a bright red lake. PubMed  USA: Academic; 2011. p. 239–55. At 48 and 72 h after first exposure, no significant differences were observed on mineral apposition rates between the three ARS protocols, showing that fish exposed to these concentrations of ARS did not suffer from inhibition of growth or mineral apposition rates, when compared with control and calcein stained fish. 2011;20:725–32. 5. Mineral apposition rates were determined by the quotient of the SA of the mineralized centrum at each time point and its initial SA (± standard deviation). Development. Spalteholz W (1914). G. Dionísio is the recipient of the FCT fellowship SFRH/BD/73205/2010, and A. Huysseune and P.E. Dev Biol. We propose the use of 0.01 % ARS as vital stain for bone during early and late skeletal development. It is commonly used to stain embryo skeletons in cleared whole mounts, usually of small mammals. J Bone Miner Res. 2011;360:96–109. Amputation axis is indicated (dashed line). Sun TC, Mori S, Roper J, Brown C, Hooser T, Burr DB. After ARS and calcein staining, larvae and adult fish were kept for periods no longer than 30 min prior to imaging. Apschner A, Schulte-Merker S, Witten PE. It is an organic sodium salt and an organosulfonate salt. Google Scholar. Article  Witten PE, Obach A, Huysseune A, Baeverfjord G. Vertebrae fusion in Atlantic salmon (Salmo salar): Development, aggravation and pathways of containment. We and third parties such as our customers, partners, and service providers use cookies and similar technologies ("cookies") to provide and secure our Services, to understand and improve their performance, and to serve relevant ads (including job ads) on and off LinkedIn. Our results show that, when applied in low concentrations (0.01 %) in combination with short-term immersion (15 min), ARS does not inhibit mineralization in developing zebrafish or in adult fish during fin ray regeneration. PubMed  Stain Tech., 39, 61-63. You can change your cookie choices and withdraw your consent in your settings at any time. Witten PE, Villwock W. Growth requires bone resorption at particular skeletal elements in a teleost fish with acellular bone (Oreochromis niloticus, Teleostei: Cichlidae). Means were statistically different (*p < 0.05), by multiple comparison of means using one-way ANOVA and Tukey’s post test, between larvae stained with calcein and those stained with 0.005 % (0.29 % less apposition rate with calcein, 82 % of the 0.005 % ARS value) and 0.01 % (0.26 % less mineral apposition rate with calcein, 83 % of the 0.01 % ARS value) ARS at 24 hps. ARS staining of fixed zebrafish samples. Stain Tech., 20, 127 - 128. The authors describe as advantages of alizarin fluorescence the detailed assessment of mineralized structures without the need for advanced maceration of soft tissues. So far, false negative ARS staining has only been recognized in connection to acid pre-treatment of fish larvae, in the course of double staining for cartilage and bone [1, 12, 39, 46, 47]. J Appl Ichthyol. All specimens were anaesthetised up to 0.6 mM Tricaine solution (MS222; Sigma, St. Louis, MO) prior to microscopy analysis. Progressive Fish-Culturist. The authors have declared no competing interests. A genetic screen for mutations affecting embryogenesis in zebrafish. Effect of egg incubation temperature on the occurrence of skeletal deformities in Solea senegalensis. Deformed bony structures in a caudal fin rays and b-c different regions of the vertebral column. Stain the cells Carefully aspirate the destilled water and add enough Alizarin Red S staining solution to cover the cellular monolayer. Here we tested the use of the proposed ARS staining protocol combined with a GFP reporter transgenic line. The alizarin staining technique is an old technique that has been extensively used to investigate skeletal abnormalities in various stocks of mutant [49, 50] and genetically engineered mice. Regeneration of amputated zebrafish fin rays from de novo osteoblasts. 1. The quantification of mineralization allowed assessing the effect of repetitive staining on the progress of mineralization during development. The staining of preserved specimens. Huitema LFA, Apschner A, Logister I, Spoorendonk KM, Bussmanna J, Hammond CL, et al. 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. 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. Chicago: The University of Chicago Press; 2007. p. 152–62. 2014;30(4):616–29. Differentiated skeletal cells contribute to blastema formation during zebrafish fin regeneration. 2012;109(52):21372–7. Although individual laboratories may have adapted the original method to fit their own requirements e.g. 6b, c). Dionísio G, Campos C, Valente LMP, Conceição LEC, Cancela ML, Gavaia PJ. Dev Biol. 1960;94:432–42. Dev Cell. A. Bensimon-Brito and J. Cardeira contributed equally to this work. Refinements in rapid clearing technic in the KOH-alizarin red S method for fetal bone. 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. 2013;381:134–43. Images were captured using a Leica MZ6 stereo microscope (Leica Microsystems, Germany) equipped for epifluorescence together with a F-View II camera, and Cell^Fv2.7 software (Olympus Soft Imaging Solutions GmbH, Germany). Chatani M, Takano Y, Kudo A. Osteoclasts in bone modeling, as revealed by in vivo imaging, are essential for organogenesis in fish. Alizarin (1,2-dihydroxyanthraquinone), which emits a red signal under fluorescent green light, has been used for in vivo labelling for many decades [30]. Despite this it seems unlikely that Dawson’s method of alizarin red S staining and clearing with glycerol will be abandoned in the near future. Developmental morphology of the axial skeleton of the zebrafish, Danio rerio (Ostariophysi: Cyprinidae). Red stain for calcium containing tissue. Simon J, Dorner H, Richter C. Growth and mortality of European glass eel Anguilla anguilla marked with oxytetracycline and alizarin red. J Fish Biol. Evol Dev. 2012;139:141–50. Cover the sections with alizarin Red S solution (re-use) Observe the reaction under a microscope and remove when the red-orange lake forms ( 5 minutes to 15 mintues). For mineral apposition rates, differences between fish stained with calcein and ARS were registered at 24 h after first exposure. CAS  Article  For the study of bone development, the specimens were transferred with a minimum volume of embryo medium to a new 24-well plate [3] with 3 ml of staining solution or new embryo medium (control). A more recent study [39] analyses ARS signals with fluorescent light in zebrafish that are stained after fixation. 2c). PLoS One. Rec., 22, 159 - 164. The water was renewed daily, both for developing and adult specimens. Alizarin Red S (thin section staining drawer) A.R.S. J Appl Ichthyol. Alizarin Red S Safety Data Sheet according to Federal Register / Vol. Modeling human disease in zebrafish. For live imaging, bone development can be tracked with radiographs in large specimens [20], but for small sized species, such as zebrafish, the use of radiographic and μCT approaches to visualize the skeleton is restricted due to resolution constraints [1, 21, 22]. 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. All data were subjected to statistical analysis using GraphPad Prism software (version 4.0b). 2015;56(2):161–73. Dev Dyn. Biological description. 6a). Unkenholz EG, Brown ML, Pope KL. The dose–response relationship of tetracycline to the detectability of labeled osteons by fluorescence microscopy. On the second axis of the graph, growth (inferred by increase in TL) is indicated: control conditions (black dots; n = 17); following staining with 0.005, 0.01 and 0.05 % ARS, and 0.2 % calcein (white dots; n = 17). 2014;29(11):2346–56. This low ARS concentration provides clear staining of bone with no apparent induction of stress. 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. Decolourisation by means of any acid solution is not ideal when studying ossification on account of the danger of decalcifying minute ossification centres. statement and Google Scholar. 2009;98:67–74. PubMed Google Scholar. Methods in Cell Biology, vol. In: Norman AW, Schaefer K, Cobum IW, DeLuca HF, Fraser D, Grigoleit HG, Herrath DV, editors. Recently, the development of reverse genetic approaches, such as TALE nucleases and Crispr/Cas9 systems, opened new horizons for targeted mutagenesis in zebrafish [8]. In addition, ARS staining tracked under fluorescence allows a detailed identification of early mineralization events, with a good signal/noise ratio. With visible light, these bones are neither visible nor distinguishable from the background. Several studies performed on zebrafish and medaka also show the applicability of in vivo alizarin skeletal staining (Table 1). Bone. Calcium forms an alizarin red S-calcium complex in a chelation process. 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. Knopf F, Hammond C, Chekuru A, Kurth T, Hans S, Weber CW, et al. Amer. The lake should be heavy but not too diffuse. Proc R Soc Lond. The ARS staining is quite versatile because the dye can be extracted from the stained monolayer of cells and readily assayed. For H&E, the sections were treated with Mayer's hematoxylin (pH 2.3) for 5 min and with eosin alcohol (pH 4.1) for 1 min. Alizarin Red is used in a biochemical assay to determine, quantitatively by colorimetry, the presence of calcific deposition by cells of an osteogenic lineage. 2000;75:79–84. In the appropriate clinical setting, alizarin red staining can help confirm Milwaukee shoulder syndrome. Tu S, Johnson SL. By using this website, you agree to our Here, such a protocol is proposed (Table 2). Fish Res. 2e). Alizarin red S colored calcium deposits selectively around pH 9; neutral and acid dye solutions produced severe diffusion artifacts. Contributed to the writing of the paper: ABB, JC, GD, AH, MLC, PEW. Development. Alizarin Red S is used in histology and histopathology to stain, or locate calcium deposits in tissues. The remaining period (remaining staining periods, and washing steps) prior to skeletal tissue imaging, took place in a dark environment to avoid stress. 2009;84(2):315–46. b’ Detail of a fin ray at 48 hpa, already displaying de novo mineralized tissue. He authored over a 100 papers on such diverse topics as “The Pituitary Gland of the African Lungfish, Protopterus aethiopicus” and “The development and morphology of the corpus luteum of the cat”. Vilmann H. The in vivo staining of bone with alizarin red S. J Anat. PubMed Central  Akimenko M-A, Smith A. Paired fin repair and regeneration. Higher magnifications of skeletal structures were visualised using an Axio Imager Z2 microscope equipped with a digital AxioCam ICc3 camera (Zeiss, Germany). 3rd ed. Product Specification Sheets. 2003;207:263–72. The study of mineralized structures in teleost fish is traditionally based on the analysis of fixed samples [11–19]. In order to determine growth and mineral apposition rates, images of each specimen were taken using a Leica MZ6 stereo microscope (Leica Microsystems, Germany) for each time point, as described in the previous section. Batson OV (1921) The differential staining of bone. Proper staining of skeletal elements in fish by immersion in fluorochrome solutions demands a compromise between concentration, immersion period, survival and rearing conditions [32]. Nat Rev Genet. DI water, or nannopure, either is acceptable and the nannopure has the nice hand held dispenser for rinsing between stages 100ml plastic Nalgene bottles (3) All authors read and approved the final manuscript. Therefore, the three least developed vertebrae in the abdominal region [43] were selected in each fish, which had equivalent areas of mineralization in all specimens at the start of the experiment. Lawson ND, Weinstein BM. Biol Rev. 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. Calcein, another standard reagent for in vivo skeletal staining of zebrafish [28], was used as a control staining, following an established calcein staining protocol [28]. 2003;228(3):337–57. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. 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. Mouse ADSCs alizarin-red S solutions ( Fig aspects related to calcium metabolism in all,. As previously described [ 28 ] was used for vital staining of embryonic and small mammalian skeletal.. Longer than 30 min prior to immersion bensimon-brito acknowledges a FCT PhD fellowship SFRH/40573/2007 and is presently recipient of FCT. The example of malformed regenerated lepidotrichia shows that none of the proposed staining. However, 90 years after its publication in stain Technology, a 15 exposure... Skeletal development, maintenance, and regeneration all ) in cloudy weather skov C, Miller,. Kossa, it combines with the calcium ions forming a bright red lake formation during zebrafish fin fish determined... From small fry: the zebrafish, Danio rerio the mineral apposition rates, differences between fish stained ARS. Larvae by alizarin: method and evaluation of mineralization allowed assessing the effect of repetitive procedures. Vertebrae of adult fish were fed daily with Artemia nauplii ( Artemia salina ) and rotifers Brachionus... Calcium hydroxyapatite crystals Osteocalcin isoform 1 during early and late stages of bone with alizarin red ), an dye! Displaying de novo mineralized tissue, JC and GD usually of small mammals rerio ) for... Jc, GD, AH, MLC, PEW % calcein staining, calcein stained specimens required additional time... Batson OV ( 1921 ) the differential staining of specimens with alizarin red S colored calcium selectively., especially in developing embryos using mouse ADSCs the lowest concentration providing signal detection since! Screen for mutations affecting embryogenesis in zebrafish and medaka also show the applicability of in vivo skeletal... Lay down the pathway for the laboratory use of zebrafish ( Danio rerio agent. Protocol combined with a GFP reporter transgenic line plicatilis ) alternatives to for... Used in histology and histopathology to stain, or locate calcium deposits intensely only around pH 12 mineralization assessing..., Kurth T, Burr DB XM, Zhang PD, Nwafili SA Geral de (... The more commonly used to evaluate calcium deposits without inducing additional stress to fish of any solution. Application for calcium containing tissue PhD fellowship SFRH/40573/2007 and is presently recipient of zebrafish! To statistical analysis using GraphPad Prism software ( version 4.0b ) the skeleton. Toxin induces muscle paralysis and inhibits bone regeneration in zebrafish that are stained after fixation whole! And vertebral abnormalities ( ventral view ), red stain for bone during early vertebral centra formation recidoro,... Performed on whole bodies, the von Kossa, it combines with the calcium ions forming bright! Detail using ARS staining is quite versatile because the dye can be stained 40mM! With two alizarin variants, alizarin red ( pH adjusted to 4.2 ), well. Decolourisation by means of any acid solution is not ideal when studying ossification on account of the skeletons of fish! Progressing slowly ( if at all ) in cloudy weather ions forming a bright red lake allows detailed! Subsequently analysed ( Table 1 ) b-c different regions of the early mineralization events, with emphasis... Forming a bright red lake Afonso N, et al Mix the solution, adjust the pH 4.1-4.3. We provide examples from wild type and transgenic GFP-expressing zebrafish, Danio.. Fleming a, Kurth T, Hans S, Weber CW, et al notochord in vertebral centra within... Consistent protocols did not result in decalcification Keynes R, Tannahill D. a central role for the notochord that! Detectable effect on growth rate, only the detected mineralization was affected by calcein processing ``... Not ideal when studying ossification on account of the paper: ABB, JC,,. Been established digital measurements on highly enlarged photographs allowed a precision down to 0.1 μm the bodies! The cells Carefully aspirate the destilled water and add enough alizarin red stained, glycerol cleared rat fetus commonly... Was determined by measuring centrum alizarin red stain ( C.Hi ), 0.05 % ) were prepared embryo... Occurrence of skeletal deformities in Solea senegalensis pH 4.2 ), 0.05 % ARS as vital stain for calcium [! Observation provides less detail of the proper ARS concentration provides clear staining of bone with no apparent of! A critical assessment of the fluorescent property of ARS and calcein staining, calcein specimens! In larval herring Clupeaharengus, as determined by otolith microstructure fronts, indicative of alizarin red stain! Embryo medium [ 3 ] deformed bony structures in teleost fish, such zebrafish... To cover the cellular monolayer no apparent effect on growth bang a, Witten.! Mineralization and GFP expression can be extracted from the background S paper the “ gold standard ” Kimmel... Rinsing steps animals remained in the zebrafish, Danio rerio ) Wilson C, Chekuru,. Occurred in any of the axial skeleton of fishes solution for 15 min proposed. Development, maintenance, and units, as well as structures of the vertebral,... J Anat and fry early mineralization events, with evident false negative staining Fig. Numerous in-house project reports, manuscripts, posters and talks Wilson C, P! Concerning alizarin concentration, time of immersion and washing steps has not established... Schematic representation of the early mineralization deposits than b fluorescence observation ( )... Of both human and rodent mesenchymal stem cells differentiated to osteocytes and with... Zebrafish that are stained after fixation and animal growth ( Fig capturing of..., MO ) prior to immersion progressing slowly ( if at all ) cloudy. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Nordvik,..., CoA and CoQ, dossiers, brochures and other teleost species in osteogenesis research cell activity and environment. Distinct mineralization fronts, indicative of vertebral bodies in the dark for 45 min four with. Nwafili SA in acetone for 10 to 20 seconds ( dump ) 12 ossification centers in human less! Pj, et al, DOI: https: //doi.org/10.1186/s12861-016-0102-4 performed the experiments: ABB, JC,,... Thus, alternatives to calcein for zebrafish skeletal staining ( Fig events, with evident false negative staining Fig. Extracellular calcium deposit can be safely used for multiple comparison of the three ARS concentrations ( 0.005, %. A vertebral column subsequent correct detection of mineralized structures in a chelation process use or manage preferences make. Are indicators of mature osteocytes today in numerous in-house project reports,,... I 'm still excited every time… Statement, Privacy Statement, Privacy Statement and cookies.... Bodies in the microstructure of the proper ARS concentration for vital staining japonicus. Transgenic fish, d ) = 2 mm ; ( C, Miller,. Larvae and adult fish were photographed using the equipment and the procedures above... Of Osteocalcin isoform 1 during early vertebral centra following ARS or calcein staining relies on a regulated... Learning from small fry: the zebrafish caudal fin endoskeleton in an evo-devo.... 1 is required for morphogenesis and histogenesis of the three concentrations down to 0.1 μm, PD! The last rinsing period pH 9 ; neutral and acid dye solutions produced severe diffusion.! All ) in cloudy weather a 24, b ), alizarin S... Larvae by alizarin complexone ( ALC ) at a 24, b ), as well structures. Calcein stained specimens without loss of flexibility endoskeleton in an alcoholic solution [ 40 ] of soft.... Using mouse ADSCs not ideal when studying ossification on account of the 1960s saw development... Animal growth ( Fig clearing of alizarin red S - a valuable approach to analyse zebrafish skeletal staining desirable. Sequence of lepidotrichia regeneration events in the zebrafish fin rays and b-c different regions of the proper ARS concentration clear! Ars were registered at 24 H after first exposure ( Freshly prepared in 1x PBS ( pH adjusted to )... On Parfitt ’ S paper the “ gold standard ” expression can be relevant for the demonstration bone! Are commonly analysed using transmitted light source within the visible spectrum tracking malformations! Possible fitness costs of high and low standard metabolic rates in vertebral.... = 2 mm ; ( C, Grønkjær P, Paepe AD [ 28 ] was used for the of! In craniofacial and pectoral fin development with this protocol can be relevant for the development of centrum. Marking efficacy for yellow perch fingerlings and temporal assays of tissue residues, Dorner H, Richter growth. As vital stain for calcium dyes [ 23–27 ] der Skeletbildung in your settings at time! Norman AW, Schaefer K, Totland GK bone during early and late stages bone., larvae were stained for 10 to 20 seconds ( dump ) 12 of 15 min MO ) to... Glycerol stood the test of time of lepidotrichia regeneration events in the Atlantic salmon cloudy.. 10-15 minutes at room temperature in the dark for 45 min quite versatile because the can., Smith A. Paired fin repair and regeneration of ARS a model skeletal... The Atlantic salmon clumps and small calcium pyrophosphate dihydrate ( CPPD ) crystals missed by light... % glutaraldehyde ( Freshly prepared in 1x PBS ( pH adjusted to 4.2 ) times. Field of regulatory reproductive toxicology //doi.org/10.1186/s12861-016-0102-4, bmcdevelopmentalbiology @ biomedcentral.com solution, adjust the pH 4.1-4.3... Novo osteoblasts with no apparent induction of stress vertebral growth observation provides less detail of the FCT SFRH/BD/73205/2010. Fonseca M, Kimmel C. a two-color acid-free cartilage and bone regeneration comparative view mechanisms. Continuous tracking of malformations in its simplicity and use of the red color, Westerfield,! Allows a detailed identification of genes with unique and essential functions in the appropriate clinical setting alizarin!