This work was supported by the National Natural Science Foundation of China (81872646; 81811540034; 81573173) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

All animal procedures outlined here have been reviewed and approved by the Animal Care Institute of The Ethics Committee of Soochow University.
1. Fish husbandry and embryo collection 14
<ol>
	<li>Feed fish three times every day; ensure the zebrafish are maintained at 28.5 &#177; 0.5 &#176;C with a 14:10 h light/dark cycle.</li>
	<li>Separate the male and female adult fish by isolation boards in spawning tanks at a 2:1 male to female ratio in the evening.</li>
	<li>The next morning, remove the isolation boards at 9:00 AM and collect the embryos 2 h later.</li>
	<li>Place the embryos in a biochemical incubator maintained at 28.5 &#176;C before the experiment.</li>
</ol>
2. Chemical exposure
<ol>
	<li>Prepare the mother stock: weigh lead acetate trihydrate (5 g), and dissolve it in ultrapure water (50 mL) with stirring. 
	CAUTION: PbAc is toxic. Wear suitable dust masks, protective clothing, and gloves. Perform the experiment under a fume hood.</li>
	<li>Select and randomly distribute zebrafish embryos into clean 6-well plates (30 embryos per well in 3 mL of zebrafish breeding water; see Table 1 for its composition). Treat the embryos with PbAc (0, 5, 10, 20 mg/L) from 2 to 120 h post fertilization (hpf). 
	NOTE: The concentrations of PbAc were chosen according to the dose-range-finding experiments. The results showed that the LC50 of lead acetate on zebrafish embryos was 41.044 mg/L at 120 hpf. Hence, the highest concentration was set to half of the LC50 and a series of solutions prepared by 2-fold dilution.</li>
	<li>Feed the larvae twice a day from 5 days post fertilization (dpf). Maintain the zebrafish embryos at 28.5 &#177; 0.5 &#176;C with a 14:10 h light/dark cycle.</li>
	<li>Refresh the Pb-free medium (zebrafish breeding water) every 24 h until 9 dpf. 
	&#8203;NOTE: Upon completion of the experiments, all lead-containing solutions were poured into a designated liquid tank and treated by the experiment management center.</li>
</ol>
3. Alizarin red staining
NOTE: Wear suitable dust masks, protective clothing, and gloves during the entire staining process. The compositions of all solutions are shown in Table 1.
<ol>
	<li>Specific staining steps 
	NOTE: The dyeing process was carried out in a 24-well plate at room temperature. After each solution was added, place the 24-well plate on the shaking table set at a low speed.
	<ol>
		<li>Remove 10 zebrafish larvae randomly from each group at 9 dpf and fix the fish in 1 mL of 2% paraformaldehyde/1x phosphate-buffered saline for 2 h.</li>
		<li>Decant the solution and wash the zebrafish larvae with 100 mM Tris-HCl (pH 7.5)/10 mM MgCl2 for 10 min.</li>
		<li>Decant the solution and incubate the larvae in the following solutions for 5 min each for destaining: anhydrous ethanol (EtOH) solution (80% EtOH/100 mM Tris-HCl (pH 7.5)/10 mM MgCl2, 50% EtOH/100 mM Tris-HCl (pH=7.5), and 25% EtOH/100mM Tris-HCl (pH=7.5).</li>
		<li>Decant the solution, remove the pigment by bleaching with a solution consisting of 3% H2O2 and 0.5% KOH, and observe once every 10 min until the pigment is completely removed.</li>
		<li>Rinse the zebrafish larvae several times with 25% glycerin/0.1% KOH for 10 min each until there are no bubbles. 
		NOTE: It is important to avoid bubbles; otherwise, the bubbles in the zebrafish body will appear as a black field of view under the microscope, which will affect observations and quantitative analysis.</li>
		<li>Stain the larvae by soaking in 1 mL of 0.01% alizarin for 50 min.</li>
		<li>Decant the solution and add 1mL of 50% glycerin/0.1% KOH for 10 min to clear the background. Store the fishes in fresh 50% glycerin/0.1% KOH for subsequent observation.</li>
	</ol>
	</li>
</ol>
4. Image acquisition
<ol>
	<li>Transfer one larva onto the glass slide each time, keeping the larva in the middle of the liquid drop.</li>
	<li>Observe the larvae under a stereo microscope.</li>
	<li>Turn on the camera, open the software (see the Table of Materials), and keep the default settings.</li>
	<li>Click on AE, and choose an appropriate exposure time (60 ms in this experiment) to obtain the best image.</li>
	<li>Capture all the images under the same settings. Save the images in .tif format for later analysis.</li>
</ol>
5. Image analysis
NOTE: See the Supplemental file for an example with a set of sample graphs for image analysis.
<ol>
	<li>Double-click the ImageJ icon and analyze the images saved in step 4.5.
	<ol>
		<li>Click on File | Open to open the images saved in step 4.5.</li>
		<li>Click on Image | Type, select 8-bit.</li>
		<li>Click on Edit | Invert.</li>
		<li>Click on Analyze | Calibrate, select Uncalibrated OD in the popup interface, check Global calibration at the bottom left of the lower interface, and click OK.</li>
		<li>Click on Analyze | Set Scale | Click to Remove scale in the popup interface, check Global below, and click OK.</li>
		<li>Click on Analyze | Set Measurements, select the item area in the popup interface, check the Limit to threshold below (to measure only the selected range), and click OK.</li>
		<li>Click on Image | Adjust| Threshold, slide the slider in the middle of the popup interface to select the appropriate threshold (change the threshold for each image) so that all targets to be tested in one image are selected, and click Set.</li>
		<li>Click on Analyze | Measure. Record the dates of each group.</li>
	</ol>
	</li>
	<li>Use one-way ANOVA followed by Tukey&#39;s multiple comparison test to analyze the differences, and set the significance level at p &#60; 0.001 (***).</li>
</ol>

<ol><li>Compston, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recommendations+for+the+registration+of+agents+for+prevention+and+treatment+of+glucocorticoid-induced+osteoporosis%3A+an+update+from+the+Group+for+the+Respect+of+Ethics+and+Excellence+in+Science%5BTitle%5D)+AND+%22Osteoporosis+International%22%5BJournal%5D)">Recommendations for the registration of agents for prevention and treatment of glucocorticoid-induced osteoporosis: an update from the Group for the Respect of Ethics and Excellence in Science.</a> Osteoporosis International. 19 (9), 1247-1250 (2008).</li>
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<li>Morin, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differences+in+fracture+prevalence+and+in+bone+mineral+density+between+Chinese+and+White+Canadians%3A+the+Canadian+Multicentre+Osteoporosis+Study+%28CaMos%29%5BTitle%5D)+AND+%22Archives+of+Osteoporosis%22%5BJournal%5D)">Differences in fracture prevalence and in bone mineral density between Chinese and White Canadians: the Canadian Multicentre Osteoporosis Study (CaMos).</a> Archives of Osteoporosis. 15 (1), 147(2020).</li>
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<li>Theppeang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Associations+of+bone+mineral+density+and+lead+levels+in+blood%2C+tibia%2C+and+patella+in+urban-dwelling+women%5BTitle%5D)+AND+%22Environmental+Health+Perspectives%22%5BJournal%5D)">Associations of bone mineral density and lead levels in blood, tibia, and patella in urban-dwelling women.</a> Environmental Health Perspectives. 116 (6), 784-790 (2008).</li>
<li>Sun, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osteoporosis+in+a+Chinese+population+due+to+occupational+exposure+to+lead%5BTitle%5D)+AND+%22American+Journal+Industrial+Medicine%22%5BJournal%5D)">Osteoporosis in a Chinese population due to occupational exposure to lead.</a> American Journal Industrial Medicine. 51 (6), 436-442 (2008).</li>
<li>Guadalupe-Grau, A., Fuentes, T., Guerra, B., Calbet, J. A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exercise+and+bone+mass+in+adults%5BTitle%5D)+AND+%22Sports+Medicine%22%5BJournal%5D)">Exercise and bone mass in adults.</a> Sports Medicine. 39 (6), 439-468 (2009).</li>
<li>Ottani, V., Raspanti, M., Ruggeri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Collagen+structure+and+functional+implications%5BTitle%5D)+AND+%22Micron%22%5BJournal%5D)">Collagen structure and functional implications.</a> Micron. 32 (3), 251-260 (2001).</li>
<li>Kelly, W. L., Bryden, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modified+differential+stain+for+cartilage+and+bone+in+whole+mount+preparations+of+mammalian+fetuses+and+small+vertebrates%5BTitle%5D)+AND+%22Stain+Technology%22%5BJournal%5D)">A modified differential stain for cartilage and bone in whole mount preparations of mammalian fetuses and small vertebrates.</a> Stain Technology. 58 (3), 131-134 (1983).</li>
<li>Barrett, R., Chappell, C., Quick, M., Fleming, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid%2C+high+content%2C+in+vivo+model+of+glucocorticoid-induced+osteoporosis%5BTitle%5D)+AND+%22Biotechnology+Journal%22%5BJournal%5D)">A rapid, high content, in vivo model of glucocorticoid-induced osteoporosis.</a> Biotechnology Journal. 1 (6), 651-655 (2006).</li>
<li>Howe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li>
<li>Fernandez, I., Gavaia, P. J., Laize, V., Cancela, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fish+as+a+model+to+assess+chemical+toxicity+in+bone%5BTitle%5D)+AND+%22Aquatic+Toxicology%22%5BJournal%5D)">Fish as a model to assess chemical toxicity in bone.</a> Aquatic Toxicology. 194, 208-226 (2018).</li>
<li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+GH%2FIGF+axis+in+arsenite-induced+developmental+toxicity+in+zebrafish+embryos%5BTitle%5D)+AND+%22Ecotoxicology+Environmental+Safety%22%5BJournal%5D)">Role of GH/IGF axis in arsenite-induced developmental toxicity in zebrafish embryos.</a> Ecotoxicology Environmental Safety. 201, 110820(2020).</li>
<li>Bergen, D. J. M., Kague, E., Hammond, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+as+an+emerging+model+for+osteoporosis%3A+a+primary+testing+platform+for+screening+new+osteo-active+compounds%5BTitle%5D)+AND+%22Frontiers+in+Endocrinology%22%5BJournal%5D)">Zebrafish as an emerging model for osteoporosis: a primary testing platform for screening new osteo-active compounds.</a> Frontiers in Endocrinology. 10, 6(2019).</li>
<li>Charles, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Utility+of+quantitative+micro-computed+tomographic+analysis+in+zebrafish+to+define+gene+function+during+skeletogenesis%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">Utility of quantitative micro-computed tomographic analysis in zebrafish to define gene function during skeletogenesis.</a> Bone. 101, 162-171 (2017).</li>
<li>Hammond, C. L., Moro, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+transgenic+reporters+to+visualize+bone+and+cartilage+signaling+during+development+in+vivo%5BTitle%5D)+AND+%22Frontiers+in+Endocrinology%22%5BJournal%5D)">Using transgenic reporters to visualize bone and cartilage signaling during development in vivo.</a> Frontiers in Endocrinology. 3, 91(2012).</li>
<li>Bensimon-Brito, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Revisiting+in+vivo+staining+with+alizarin+red+S--a+valuable+approach+to+analyse+zebrafish+skeletal+mineralization+during+development+and+regeneration%5BTitle%5D)+AND+%22BMC+Developmental+Biology%22%5BJournal%5D)">Revisiting in vivo staining with alizarin red S--a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration.</a> BMC Developmental Biology. 16, 2(2016).</li>
</ol>

The authors have no conflicts of interest to disclose.

The zebrafish is a suitable model for studying bone metabolic disease. Compared to rodent models, zebrafish models are relatively fast to establish, and measurement of the severity of disease is easier. In wild-type zebrafish larvae, mineralization of the head skeleton occurs at 5 dpf and the axial skeleton at 7 dpf15. Thus, cranial bones such as PS, OP, CB, and NC are well developed at 9 dpf. After the larvae were completely destained and bleached, the soft tissues were cleared, resulting in a transparent appearance of the fish body. The alizarin red staining reagent was added to stain and visualize the mineral bones in zebrafish in red. 
Image analysis is critical to obtain reliable experimental conclusions in this experiment. Photographs of zebrafish with good posture and clean background were selected for quantitative analysis. When we quantify the stained area for a single image, all mineralized bones stained in red of one fish will be calculated. Thus, we can compare the bone mass changes between the lead-exposed and the control groups. In this study, PbAc exposure caused developmental toxicity in zebrafish, and a significant reduction in the stained area of mineral bone was observed in the 10 and 20 mg/L PbAc-exposure groups at 9 dpf. Thus, early embryonic exposure to PbAc reduced the bone mass of zebrafish larvae. Figure 1 shows PbAc-induced bone loss visualized by alizarin red staining in the zebrafish larvae. 
Dyes that bind to calcified matrix are used to label the entire skeleton. Calcein is a fluorescent chromophore that can also specifically bind to calcium in live tissue and has been used to label bone structures and study bone growth10. Unlike calcein, alizarin red staining of fixed tissue generates a permanent record of skeletal changes that can facilitate comparisons of several specimens. Microcomputed tomography (Micro CT) can provide accurate quantitative analysis of mineralized tissue by acquiring a series of 2D X-rays. However, because of the small size of zebrafish and many of the bones of the developing zebrafish skeleton being thin, Micro CT analysis tools cannot accurately characterize these bones16.
Fluorescent transgenic reporter lines also help visualize skeletal development in live larvae or even more mature fish in real time17. Similarly, alizarin red S in vivo staining permits the evaluation of live fish and the continuous tracking of malformations18. Thus, alizarin red staining is a useful and cost-effective way to analyze bone loss in zebrafish larvae. However, because of the complexity of the experimental steps and the number of solutions used, the final results of the analysis of alizarin red-stained images may be affected by the experimental operation. Further, it is difficult to use this staining method for adult zebrafish due to increased body volume and soft tissues; Micro CT analysis or transgenic lines would be a better choice for skeletal imaging of adult zebrafish. In summary, the protocol presented here can be used to study the changes in bone mineralization in zebrafish larvae following chemical toxicant exposure. This procedure could be useful to establish a zebrafish model to study bone disease and develop new therapeutic drugs.

Recent studies have confirmed that osteoporosis due to glucocorticoids, aromatase inhibitors, and excessive alcohol consumption is common1,2. Lead (Pb) is a toxic metal found in plants, soil, and aquatic environments3. Although the adverse effects of Pb on the human body have attracted much attention, its irreversible impact on bone needs to be investigated further. Lead intoxication causes a diverse array of pathological changes in both the developing and adult skeleton, affecting normal life activities. Studies have found an association between chronic Pb exposure and bone damage4, including impaired bone structures5,6, reduced bone mineral density, and even increased risk of osteoporosis7.
Mineralized tissue is of great importance to bone strength8, and bone mineralization matrix deposition is a critical index of bone formation9. Alizarin red has a high affinity for calcium ions, and alizarin red staining is a standard procedure for assessing bone formation10. According to this method, mineralized tissue is stained red, while all other tissue remains transparent. The stained area is then quantified by digital image analysis11.
Zebrafish is an important model organism widely used in drug discovery and disease models. Genetic studies in zebrafish and humans have demonstrated similarities in the underlying mechanisms of skeletal morphogenesis at the molecular level12. Moreover, high-throughput drug or biomolecule screening is more feasible in large clutches of zebrafish than murine models, facilitating the mechanistic study of proosteogenic or osteotoxic molecules13. Differential staining of the skeleton in toto10 is frequently used in studying skeletal dysplasia in small vertebrates and mammalian fetuses. Alizarin red staining was performed to investigate the bone developmental toxicity of chemicals in zebrafish larvae. Herein, we used lead as an example to describe a protocol for detecting lead acetate-induced bone defects in zebrafish larvae.

<table><tbody><tr><td>1 M Tris-HCl (pH=7.5)</td><td>Solarbio,Beijing,China</td><td>21</td><td>for detaining</td></tr><tr><td>4% Paraformaldehyde Fix Solution</td><td>BBI,Shanghai,China</td><td>14</td><td>fixing tissues</td></tr><tr><td>10x PBS buffer</td><td>BBI,Shanghai,China</td><td>15</td><td>for fixing</td></tr><tr><td>35% H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Yonghua,Jiangsu,China</td><td>8</td><td>removing pigment</td></tr><tr><td>50 mL Centrifuge tube</td><td>AKX,Jiangsu,China</td><td>4</td><td></td></tr><tr><td>95% Anhydrous ethanol</td><td>Enox,Jiangsu,China</td><td>2</td><td>destaining</td></tr><tr><td>Alizarin red (Purity 99.5%)</td><td>Solarbio,Beijing,China</td><td>1</td><td>staining</td></tr><tr><td>Biochemical incubator</td><td>Yiheng,Shanghai,China</td><td>3</td><td>raising zebrafish embryos</td></tr><tr><td>Electronic scale</td><td>Sartorius,Germany</td><td>5</td><td>weighing the solid raw materials</td></tr><tr><td>Glycerin (Purity 99.5%)</td><td>BBI,Shanghai,China</td><td>7</td><td>storing the stained fish</td></tr><tr><td>ImageJ (software)</td><td>USA</td><td>9</td><td>digital analysis</td></tr><tr><td>KOH (Purity 99.9%)</td><td>Sigma,America</td><td>10</td><td>bleaching solution</td></tr><tr><td>Lead acetate trihydrate (Purity 99.5%)</td><td>Aladdin,Shanghai,China</td><td>11</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2 &lt;/sub&gt;(Purity 99.9%)</td><td>Aladdin,Shanghai,China</td><td>12</td><td>cleaning solution</td></tr><tr><td>NIS-Elements F (software)</td><td>Nikon, Japan</td><td>13</td><td>observing and taking photos</td></tr><tr><td>Pipe</td><td>AKX, Jiangsu, China</td><td>18</td><td>removal of embryos and solution</td></tr><tr><td>plates (24-well)</td><td>Corning,America</td><td>17</td><td>container for staining embryos</td></tr><tr><td>plates (6-well)</td><td>Corning,America</td><td>16</td><td>container for breeding embryos</td></tr><tr><td>Shaking table</td><td>Beyotime, China</td><td>19</td><td>mixing the solution</td></tr><tr><td>Stereo microscope</td><td>Nikon,Japan</td><td>20</td><td>observing and taking photos</td></tr><tr><td>Zebrafish</td><td>Zebrafish Experiment Center of Soochow University,Suzhou,China</td><td>22</td><td>experimental animal</td></tr></tbody></table>

using alizarin red staining to detect chemically induced bone loss in zebrafish larvae

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the specific effects and mechanisms in zebrafish remain unclear. Alizarin red has a high affinity for calcium ions and can help visualize the bone and illustrate skeletal mineral mass. In this study, we aimed to detect lead acetate (PbAc)-induced bone loss in zebrafish larvae by using alizarin red staining. Zebrafish embryos were treated with a series of PbAc concentrations (0, 5, 10, 20 mg/L) between 2 and 120 h post fertilization. Whole-mount skeletal staining was conducted on larvae at 9 days post fertilization, and the total stained area was quantified using ImageJ software. The results indicated that the mineralized tissues were stained in red, and the stained area decreased significantly in the PbAc-exposure group, with a dose-dependent change in bone mineralization. This paper presents a staining protocol for investigating skeletal changes in PbAc-induced bone defects. The method can also be used in zebrafish larvae for the detection of bone loss induced by other chemicals.

Alizarin red staining is a sensitive and specific method for measuring changes in bone mineralization in zebrafish larvae. In this study, we have observed that PbAc had adverse effects on zebrafish larvae, including death, malformation, decreased heart rate, and body length shortening. Moreover, the mineral skeleton areas of zebrafish larvae were evaluated to examine PbAc-induced bone loss. At 9 dpf (Figure 1A), many bones of the head skeleton are mineralized and hence stained in red, such as parasphenoid (PS), opercle (OP), ceratobranchial (CB), and notochord (NC). In contrast, otoliths (OT) (non-bony structures) appear brown-black rather than red. Digital analysis was performed to quantify the total stained area in each image. Compared with the control group, lead acetate groups treated with 10 and 20 mg/L PbAc showed a significant decrease (p &#60; 0.001) in the stained area (Figure 1B). The changes in bone mineralization showed dose-dependency.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63251/63251fig01.jpg" /> 
Figure 1: Effects of different concentrations of PbAc on zebrafish larvae skull. (A) Images of the dorsal aspect of the head bone stained with alizarin red in larvae at 9 dpf. The mineralized tissue is stained in red. Scale bars = 0.5 mm. (B) Changes in relative mineralized area at 9 dpf; 10 larvae per group. Data are expressed as mean &#177; SEM. Three replicates were performed. p &#60; 0.001 ***. Abbreviations: PS = parasphenoid; OP = opercle; OT = otolith; CB = ceratobranchial; NC = notochord; dpf = days post fertilization. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63251/63251fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td colspan="2">Composition</td>
		</tr>
		<tr>
			<td>Zebrafish breeding water</td>
			<td colspan="2">pH 7-7.3, 27-29 &#176;C, conductivity 450-550 &#181;s, salinity 0.25-0.75 %0, dissolved oxygen &#62;6 mg/L, photoperiod 14/10 h, hardness 100-200 mg/L, chlorine 0 mg/L, ammonia nitrogen concentration &#60;0.02 mg/L, nitrite &#60;1 mg/L, nitrate &#60;50 mg/L, carbon dioxide &#60;50 mg/L</td>
		</tr>
		<tr>
			<td>Mixed solution (50 mL) of 2% paraformaldehyde and 1x PBS</td>
			<td colspan="2">25 mL of 4% paraformaldehyde, 5 mL of 10x PBS buffer, and double distilled water (ddH2O) q.s. to 50 mL</td>
		</tr>
		<tr>
			<td>Mixed solution (50 mL) of 100 mM Tris-HCl (pH 7.5) and 10 mM MgCl2</td>
			<td colspan="2">5 mL of 1 M Tris-HCl (pH=7.5), 0.5 mL of 1 M MgCl2, and ddH2O q.s. to 50 mL</td>
		</tr>
		<tr>
			<td>Mixed solution (50 mL) of 80% anhydrous ethanol, 10 mM MgCl2, and 100 mM Tris-HCl (pH=7.5)</td>
			<td colspan="2">42.1 mL of 95% anhydrous ethanol, 5 mL of 1 M Tris-HCl (pH=7.5), 0.5 mL of 1 M MgCl2, and ddH2O q.s. to 50 mL</td>
		</tr>
		<tr>
			<td>Mixed solution (50 mL) of 50% anhydrous ethanol and 100 mM Tris-HCl (pH=7.5)</td>
			<td colspan="2">26.3 mL of 95% anhydrous ethanol, 5 mL of 1 M Tris-HCl (pH=7.5), and ddH2O q.s. to 50 mL</td>
		</tr>
		<tr>
			<td>Mixed solution (50 mL) of 25% anhydrous ethanol and 100 mM Tris-HCl (pH=7.5)</td>
			<td colspan="2">13.2 mL of 95% anhydrous ethanol, 5 mL of 1 M Tris-HCl (pH=7.5), and ddH2O q.s. to 50 mL</td>
		</tr>
		<tr>
			<td>Mixed solution of 3% H2O2 solution and 0.5% KOH solution</td>
			<td colspan="2">Equal amounts of 6% H2O2 and 1% KOH mixed before use</td>
		</tr>
		<tr>
			<td>Mixed solution (50 mL) of 25% glycerin and 0.1% KOH</td>
			<td colspan="2">12.5 mL of 100% glycerin, 0.25 mL of 20% KOH, and ddH2O q.s. to 50 mL</td>
		</tr>
		<tr>
			<td>0.01% Alizarin (50 mL)</td>
			<td colspan="2">1 mL of 0.5% Alizarin, 12.5 mL of 100% glycerin, 5 mL of 1 M Tris-HCl (pH=7.5), and ddH2O q.s. to 50 mL</td>
		</tr>
		<tr>
			<td>Mixed solution (50 mL) of 50% glycerin and 0.1% KOH</td>
			<td colspan="2">25 mL of 100% glycerin, 0.25 mL of 20% KOH, and ddH2O q.s. to 50 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of solutions used in alizarin red staining protocol. Abbreviation:&#160;q.s. = quantum sufficient (as required).
Supplemental file: A set of sample graphs for image analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63251/SUPPLEMENTAL FILE_63251.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae at JoVE.com

Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae

Here, we have used alizarin red staining to show that lead acetate exposure causes a bone mass change in zebrafish larvae. This staining method can be adapted to the investigation of bone loss in zebrafish larvae loss induced by other hazardous toxicants.

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the ...

using-alizarin-red-staining-to-detect-chemically-induced-bone-loss

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

4.6K Views.  Medical College of Soochow University. Here, we have used alizarin red staining to show that lead acetate exposure causes a bone mass change in zebrafish larvae. This staining method can be adapted to the investigation of bone loss in zebrafish larvae loss induced by other hazardous toxicants.

Video: Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Laser Ablation Method to Study Bone Regeneration in Zebrafish Larvae

Two-Photon Laser-Mediated Ablation of Osteoblasts in Developing Zebrafish Larvae

Zebrafish (Danio rerio) have an outstanding capacity to regenerate different organs and appendages. Bone regeneration in zebrafish has been studied using different methods such as fin amputation, scale plucking, skull trepanation, and microscopic approaches. Using a confocal laser scanning setup equipped with a two-photon laser, a laser ablation method was developed as a lesion paradigm to ablate bone-forming cells (osteoblasts) in the developing opercle of zebrafish larvae. The method described here allows the ablation of cells in a precise manner, as the area, shape, and depth can be finely adjusted. In addition, this method allows imaging of the area before and just after the ablation, so that short-term effects of the injury can be analyzed. In this experimental setup, the immune response after ablation of osteoblasts in the injured area was studied. An increase in the recruitment of macrophages was observed after ablation, indicating the relevance of their presence during bone regeneration.

Author Spotlight: Immune-Mediated Bone Regeneration &#8212; The Critical Role of Macrophages and Controlled Immunomodulation in Restorative Processes

This protocol describes a two-photon laser ablation approach carried out in zebrafish larvae, which serves as a model to study bone regeneration and the effects of the immune response to ablation.

Zebrafish (Danio rerio) have an outstanding capacity to regenerate different organs and appendages. Bone regeneration in zebrafish has been studied using ...

Removing Zebrafish Scales for Culturing and Bone Biology Studies

In Vitro Culturing Technique for Studying Cellular Dynamics in Zebrafish Scales

Zebrafish scales offer a variety of advantages for use in standard laboratories for teaching and research purposes. Scales are easily collected without the need for euthanasia, regenerate within a couple of weeks, and are translucent and small, allowing them to be viewed using a standard microscope. Zebrafish scales are especially useful in educational environments, as they provide a unique opportunity for students to engage in hands-on learning experiences, particularly in understanding cellular dynamics and in vitro culturing methods. The main objective of this protocol is to describe a method for collecting and maintaining zebrafish scales in culture for use in a variety of biological studies using basic laboratory equipment. Additionally, the protocol details their use in understanding bone homeostasis by examining the activity of bone cells involved in bone resorption and deposition. It also includes additional protocols for general techniques, such as the visualization of nuclei and apoptotic cells. The in vitro culturing protocol produces reliable results with minimal reagents and equipment. This article discusses the benefits of using in vitro cultures of zebrafish scales to foster scientific inquiry and outlines the resources needed to support their integration into educational settings.

Author Spotlight: Advancing Bone Cell Activity Research with Zebrafish Scales 

This protocol offers a method to study cellular dynamics using a simple in vitro culture technique. It provides an opportunity for zebrafish researchers and educators to study cellular processes, such as those related to bone homeostasis and basic cell biology, by visualizing fluorescent nuclei and apoptotic cells within the scales.

Zebrafish scales offer a variety of advantages for use in standard laboratories for teaching and research purposes. Scales are easily collected without ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Biology

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms and pharmaceutical treatments of osteoporosis. Cathepsin K (Ctsk)-Cre mice have been widely used for functional studies of osteoclasts. The signal transducer and activator of transcription 3 (STAT3) is relevant in bone homeostasis, but its role in osteoclasts in vivo remains poorly defined. To provide the in vivo evidence that STAT3 participates in osteoclast differentiation and bone metabolism, we generated an osteoclast-specific Stat3 deletion mouse model (Stat3 fl/fl; Ctsk-Cre) and analyzed its skeletal phenotype. Micro-CT scanning and 3D reconstruction implied increased bone mass in the conditional knockout mice. H&#38;E staining, calcein and alizarin red double staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed to detect bone metabolism. In short, this protocol describes some canonical methods and techniques to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

This protocol describes a canonical method to understand the critical genes controlling osteoclast activity in vivo. This method uses a transgenic mouse model and some canonical techniques to analyze skeletal phenotype.

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms ...

Harvesting and Visualization of Elasmoid Scales of Zebrafish During In Vivo Regeneration

Zebrafish Scale Regeneration In Toto and Ex Vivo Culture of Scales

Skeletal diseases are often complex in their etiology and affect millions of people worldwide. Due to the aging population, there is a need for new therapeutics that could ease the burden on healthcare systems. As these diseases are complex, it is difficult and expensive to accurately model bone pathophysiology in a lab setting. The challenge for the field is to establish a cost-effective, biologically relevant platform for modeling bone disease that can be used to test potential therapeutic compounds. Such a platform should ideally allow dynamic visualization of cell behaviors of bone-building osteoblasts and bone-degrading osteoclasts acting in their mineralized matrix environment. Zebrafish are increasingly used as models due to the availability of genetic tools, including transgenic reporter lines, and the fact that some skeletal tissues (including the scales) remain translucent to adulthood, allowing dynamic imaging options. Since zebrafish scales have both osteoblasts and osteoclasts and are highly abundant, they provide an easily accessible and abundantly available resource of independent bone units. Moreover, once removed, adult zebrafish scales fully regenerate, therefore offering a way to study the spatiotemporal growth of mineralized tissue in vivo. Here, we detail protocols for harvesting and tracking the regeneration of the scales. Lastly, a protocol for stable culture of scales ex vivo for a week and following the healing response after controlled damage to the mineralized matrix of the scale over time is also presented.

Author Spotlight: Use of Fish Scales for Bone Remodeling Research &#8211; Advancements in Ex Vivo Imaging and Dissecting Cell Interactions

This protocol describes the harvesting and visualization of elasmoid scales of zebrafish during in vivo regeneration. In addition, the ex vivo culture of these scales for up to 7 days after harvest is presented.

Skeletal diseases are often complex in their etiology and affect millions of people worldwide. Due to the aging population, there is a need for new ...

Alizarin Red Staining: A Technique to Visualize Mineralization by Cultured Osteoblasts

Source: Kampleitner, C., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/58077">Biological Compatibility Profile on Biomaterials for Bone Regeneration.</a> J. Vis. Exp. (2018).
This video demonstrates a staining technique for detecting the extent of mineralization or calcium deposition in cultured primary osteoblasts using Alizarin Red dye.

Barwienie czerwienią alizaryny: technika wizualizacji mineralizacji przez hodowane osteoblasty

Source: Kampleitner, C., et al. Biological Compatibility Profile on Biomaterials for Bone Regeneration. J. Vis. Exp. (2018).
This video demonstrates a ...

Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae

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Przeglądaj więcej filmów

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Two-Photon Laser-Mediated Ablation of Osteoblasts in Developing Zebrafish Larvae

In Vitro Culturing Technique for Studying Cellular Dynamics in Zebrafish Scales

An Assay for Lateral Line Regeneration in Adult Zebrafish

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Zebrafish Scale Regeneration In Toto and Ex Vivo Culture of Scales

Barwienie czerwienią alizaryny: technika wizualizacji mineralizacji przez hodowane osteoblasty