Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration
- A. Bensimon-Brito†1, 2, 6Email authorView ORCID ID profile,
- J. Cardeira†1, 3,
- G. Dionísio1, 4,
- A. Huysseune2,
- M. L. Cancela1, 5 and
- P. E. Witten2
© Bensimon-Brito et al. 2016
Received: 24 August 2015
Accepted: 8 January 2016
Published: 19 January 2016
The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. 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. Alizarin compounds, either in the form of alizarin red S (ARS) or alizarin complexone (ALC), have long been used to stain the mineralized skeleton in fixed specimens from all vertebrate groups. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. There is a fundamental concern on whether ARS vital staining, achieved by adding ARS to the water, can affect bone formation in juvenile and adult zebrafish, as ARS has been shown to inhibit skeletal growth and mineralization in mammals.
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. Early and late stages of bone development are equally unaffected by this vital staining protocol. From all tested concentrations, 0.01 % ARS yielded correct detection of bone calcium deposits without inducing additional stress to fish.
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. We provide examples from wild type and transgenic GFP-expressing zebrafish, for endoskeletal development and dermal fin ray regeneration.
Skeletal mineralization relies on a tightly regulated connection between cell activity and extracellular environment. 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. 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 .
Teleost fish, such as zebrafish (Danio rerio), are recognized models to study skeletal development and regeneration . 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 . In addition, the complete genome sequence and its annotation are available, as well as a broad array of molecular and cellular tools. 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 . Recently, the development of reverse genetic approaches, such as TALE nucleases and Crispr/Cas9 systems, opened new horizons for targeted mutagenesis in zebrafish . Overall, these advantages make zebrafish a valuable vertebrate model system, widely used in fundamental and applied research (reviewed by [2, 9, 10]).
The study of mineralized structures in teleost fish is traditionally based on the analysis of fixed samples [11–19]. For live imaging, bone development can be tracked with radiographs in large specimens , 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]. 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.
Fluorescent calcium dyes (e.g., calcein, tetracycline, xylenol orange and alizarin red) can label calcium-containing tissues and be used to follow skeletal mineralization in vivo. Sclerochronology, in the frame of fish stock assessment, is a common application for calcium dyes [23–27]. 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. In addition, the fluorescence spectrum of calcein is similar to that obtained with fish tissue autofluorescence . Thus, alternatives to calcein for zebrafish skeletal staining are desirable.
Alizarin (1,2-dihydroxyanthraquinone), which emits a red signal under fluorescent green light, has been used for in vivo labelling for many decades . Vital staining of fish bone is accomplished with two Alizarin variants, Alizarin red S (ARS) and alizarin complexone (ALC). In a study on Japanese flounder Paralichthys olivaceus  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. Several studies performed on zebrafish and medaka also show the applicability of in vivo alizarin skeletal staining (Table 1). Yet, published protocols suffer from two shortcomings. First, a consistent protocol concerning alizarin concentration, time of immersion and washing steps has not been established. Second, possible negative effects of alizarin on bone growth and mineralization have not been assessed. Since alizarin has been described to inhibit growth and mineralization in vivo in rats, rabbits and guinea-pigs , a careful validation of alizarin live staining protocols is required.
Overview of studies using in vivo staining with alizarin compounds (ALC and ARS) by immersion for in vivo skeletal analysis or paraformaldehyde fixed teleost specimens. Species names, dye concentrations, duration of immersion, wash steps, and literature references are indicated
Time of immersion & washing
2–3 h / rinsing
DeLaurier et al. 2010 
O/N / rinsing
Renn et al. 2013 
n.d. / rinsing
Inohaya et al. 2007 
0.005 % + HEPES
Larvae 1–2 h; juvenile ON / rinsing
Kimmel et al. 2010 
2 h / rinsing
Willems et al. 2012 
2 h-4 h / 2 h-ON
To et al. 2012 
10 min / rinsing
Tu and Johnson 2011 
Danio rerio & Oryzias latipes
Chatani et al. 2011 
24 h / rinsed
Bashey 2004 
n.d. / rinsing 10 min
Recidoro et al. 2014 
5 min / rinsing
Huitema et al. 2012 
Fleming et al. 2004 
Eames et al. 2010 
Yan et al. 2005 
6 h / n.d.
Dougherty 2008 
24 h / n.d.
Partridge et al. 2009 
12 h / n.d.
Taylor et al. 2005 
6–24 h / n.d.
Iglesias et al. 1997 
12 h / n.d.
Bang et al. 2007 
23 h / n.d.
Skov et al. 2001 
24 h / n.d.
Lagardère et al. 2000 
24 h / n.d.
3 h / n.d.
Baer and Rosch 2008 
15 min / n.d
Nemoto et al. 2007 
24 h / 4 h
Liu et al. 2009 
24 h / 4 h
Results and discussion
Alizarin red S in vivo staining - exploring optimal concentrations
We also observed that calcein (Fig. 2d), under the established concentration , displayed a higher background staining when observed with epifluorescence compared to all ARS concentrations. To eliminate the background staining, calcein stained specimens required additional, time consuming, rinsing steps.
Next, we tested if different concentrations of ARS and calcein affected mineral apposition and animal growth (Fig. 1). We did not observe significant differences in growth rate either among ARS treated larvae, or when comparing ARS-treated, calcein-treated and control groups (Fig. 2e). This shows that none of the staining protocols has a detectable effect on growth. For mineral apposition rates, differences between fish stained with calcein and ARS were registered at 24 h after first exposure. 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. As there was no significant effect on growth rate, only the detected mineralization was affected by calcein.
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.
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. We propose the use of 0.01 % ARS as vital stain for bone during early and late skeletal development. This low ARS concentration provides clear staining of bone with no apparent induction of stress. The data on calcein staining suggest a mineralization inhibition at 24 h after first exposure, possibly due to the high concentration of the staining solution when compared with the tested ARS solutions. 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.
ARS staining of regenerating caudal fin lepidotrichia
ARS detection sensitivity in fixed specimens
Combination of ARS in vivo staining with GFP reporter lines, and a tool to reveal skeletal malformations
Bench protocol. Steps of the proposed ARS in vivo staining protocol
1. Prepare a 0.01 % ARS solution, using water from the system in which fish were previously maintained (system water or embryo medium)
1.1. A 5× concentrated solution (0.05 %) can be prepared with distilled water, then diluted in embryo medium or system water to 0.01 % working solution before use
1.2. Adjust pH to 7.4 with KOH solution
1.3. Keep solution in the dark when storing
2. Transfer fish to ARS solution
2.1. Adult specimens can be transferred with fish nets
2.2. Larval specimens can be transferred using Pasteur pipettes
3. Stain for 15 min with ARS solution
4. Rinse at least 3 times for 5 minutes in embryo medium or system water
4.1. Substitute staining solution with new embryo medium or system water, or transfer fish into new containers, as described in points 2.1. and 2.2.
5. Perform image analysis and photograph acquisition
5.1. Anaesthetize specimens with up to 0.6 mM MS222
5.2. Accommodate specimens for imaging (e.g., Petri dishes, glass-bottom dishes, excavated slides)
5.3. Use fluorescent microscope or stereomicroscope, depending on the desired magnification, coupled to the appropriate fluorescent filter
5.4. Image under green fluorescent light (510–550 nm)
6. Recover fish from anaesthesia, by transferring them to new embryo medium or system water
Ethics statement on animal experiments
Animal handling and experiments were accredited by the Portuguese Direcção Geral de Veterinária (DGV). All the experimental procedures involving animals followed the EU (Directive 2010/63/EU) and National (Decreto-Lei 113/2013) legislation for animal experimentation and welfare.
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 , with a photoperiod of 14 h light / 10 h dark. 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). During the experiments, larvae were fed daily with Artemia nauplii (Artemia salina) and rotifers (Brachionus plicatilis).
For the regeneration experiments, 3 months old adult fish were anaesthetised with 0.6 mM Tricaine (MS222; Sigma, St. Louis, MO) and caudal fin rays (lepidotrichia) were amputated one segment proximal to the first bifurcation. 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]. The fish were fed twice to satiation with commercial flakes (Benelux, Ooigem). The water was renewed daily, both for developing and adult specimens.
For fixed samples, all specimens (at 10 dpf and 30 dpf and three month old fish) were euthanized with an overdose of MS 222 and subsequently fixed for 12 h in neutral buffered 4 % paraformaldehyde. All specimens were stained for 15 min with 0.01 % ARS (3,4-Dihydroxy-9,10-dioxo-2-anthracenesulfonic acid sodium salt, from Sigma-Aldrich, St. Louis, MO) dissolved in 70 % ethanol . For a better visualization of the mineralized structures in adult fish, specimens were macerated with 3 % KOH for 12 h and subsequently dissected.
For vital staining, three ARS concentrations (0.005, 0.01 and 0.05 %) were prepared in embryo medium . The pH was adjusted to 7.4 with KOH. No precipitated ARS occurred in any of the three concentrations.
For the study of bone development, the specimens were transferred with a minimum volume of embryo medium to a new 24-well plate  with 3 ml of staining solution or new embryo medium (control). The animals remained in the staining solution for 15 min. Staining was performed once a day from 6 to 10 dpf, in each of the three ARS solutions described above. 0.2 % calcein  was used as a reference dye for mineral staining. In this case, larvae were stained for 10 min, as previously described . 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. In all cases, we assured that no dye residues were externally visible after the last rinsing period. If so, additional rinsing was conducted.
Stress levels were assessed by observing variations in the opercular movement frequency, as previously described , upon fish immersion during the first minute of staining and for 1 min at end of the staining period, before rinsing. The remaining period (remaining staining periods, and washing steps) prior to skeletal tissue imaging, took place in a dark environment to avoid stress. However, our personal observations suggest that there is no apparent effect on staining efficiency or fish health if animals remain exposed to light.
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). Adult fish were rinsed 3 times after each staining event for 5 min also in system water.
After ARS and calcein staining, larvae and adult fish were kept for periods no longer than 30 min prior to imaging. All specimens were anaesthetised up to 0.6 mM Tricaine solution (MS222; Sigma, St. Louis, MO) prior to microscopy analysis. Imaging was performed under green (510–550 nm) and blue (450–480 nm) fluorescent light to image ARS and calcein staining, respectively, and under visible light for total length (TL) measurements. 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). Higher magnifications of skeletal structures were visualised using an Axio Imager Z2 microscope equipped with a digital AxioCam ICc3 camera (Zeiss, Germany).
Tg(fli1:egfp) transgenic fish  were used to validate the suitability of ARS vital staining applied to GFP labelled fish during the regeneration of the caudal fin rays and the development of caudal vertebrae.
ARS staining was also used to detect skeletal deformities. The analysed deformities were not induced, but developed under regular rearing conditions. All fish were photographed using the equipment and the procedures described above.
Growth rate and mineral apposition in vertebral centra
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.
The TL of individual fish was determined prior to immersion. 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.
Mineral apposition rates were assessed by tracing the area of three vertebral centra in each specimen in sagittal view (Fig. 1a; anterior-posterior axis). Due to individual variability and the increasing number of mineralized vertebral bodies in different developmental stages, it was not possible to track the development of the same vertebrae in all individuals. Therefore, the three least developed vertebrae in the abdominal region  were selected in each fish, which had equivalent areas of mineralization in all specimens at the start of the experiment. Nomenclature and histomorphometric methods were based on Parfitt’s standards . On lateral microphotographs of vertebral bodies, the mineralized surface area (SA - Fig. 1b) of the centrum was determined by measuring centrum height (C.Hi) and width (C.Wi). 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).
All measurements of growth and mineral apposition rates were performed using the software ImageJ 1.47d (Wayne Rasband, National Institutes of Health, USA). Digital measurements on highly enlarged photographs allowed a precision down to 0.1 μm.
All data were subjected to statistical analysis using GraphPad Prism software (version 4.0b). One-way ANOVA was used for the analysis of variance and Tukey’s post-test was used for multiple comparison of means.
This work was funded in part by European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Program and national funds through FCT – Foundation for Science and Technology, under the project “PEst-C/MAR/LA0015/2011 and UID/Multi/04326/2013.
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. J. Cardeira is the recipient of a FCT fellowship SFRH/BD/52425/2013, within the ProRegeM PhD Programme, Department of Biomedical Sciences and Medicine from the University of Algarve. G. Dionísio is the recipient of the FCT fellowship SFRH/BD/73205/2010, and A. Huysseune and P.E. Witten acknowledge a grant from FWO 3G.0040.08.
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