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 even...

<ol>
	<li>Compston, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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><li>Rachner, T. D., Gobel, A., Jaschke, N. P., Hofbauer, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+preventing+bone+loss+induced+by+aromatase+inhibitors.">Challenges in preventing bone loss induced by aromatase inhibitors.</a> Journal of Clinical Endocrinology &amp; Metabolism. 105 (10), (2020).</li><li>Kataba, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+exposure+to+environmentally+relevant+lead+levels+induces+oxidative+stress+and+neurobehavioral+alterations+in+larval+zebrafish+(Danio+rerio).">Acute exposure to environmentally relevant lead levels induces oxidative stress and neurobehavioral alterations in larval zebrafish (Danio rerio).</a> Aquatic Toxicology. 227, 105607 (2020).</li><li>Morin, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+fracture+prevalence+and+in+bone+mineral+density+between+Chinese+and+White+Canadians:+the+Canadian+Multicentre+Osteoporosis+Study+(CaMos).">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><li>Lee, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+lead+poisoning+magnifies+bone+detrimental+effects+in+an+ovariectomized+rat+model+of+postmenopausal+osteoporosis.">Chronic lead poisoning magnifies bone detrimental effects in an ovariectomized rat model of postmenopausal osteoporosis.</a> Experimental Toxicologic Pathology. 68 (1), 47-53 (2016).</li><li>Theppeang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+of+bone+mineral+density+and+lead+levels+in+blood,+tibia,+and+patella+in+urban-dwelling+women.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoporosis+in+a+Chinese+population+due+to+occupational+exposure+to+lead.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+and+bone+mass+in+adults.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+structure+and+functional+implications.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+differential+stain+for+cartilage+and+bone+in+whole+mount+preparations+of+mammalian+fetuses+and+small+vertebrates.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+high+content,+in+vivo+model+of+glucocorticoid-induced+osteoporosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+as+a+model+to+assess+chemical+toxicity+in+bone.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+GH/IGF+axis+in+arsenite-induced+developmental+toxicity+in+zebrafish+embryos.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+an+emerging+model+for+osteoporosis:+a+primary+testing+platform+for+screening+new+osteo-active+compounds.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+quantitative+micro-computed+tomographic+analysis+in+zebrafish+to+define+gene+function+during+skeletogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+transgenic+reporters+to+visualize+bone+and+cartilage+signaling+during+development+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+in+vivo+staining+with+alizarin+red+S--a+valuable+approach+to+analyse+zebrafish+skeletal+mineralization+during+development+and+regeneration.">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 tr...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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% H2O2</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>MgCl2 (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 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 ...

This method can help detect the bone toxicity induced by chemicals in zebrafish. Alizarin Red Staining of fixed tissue generates a permanent record of skeletal changes that can facilitate comparisons of several specimens. This technique could be helpful to study osteoporosis in the zebrafish model, since one of the characteristics of osteoporosis is the decrease in bone mass.Remove 10 zebrafish larva randomly from each group at nine days post fertilization, and fix the fish in one milliliter of 2%paraform aldehyde and 1X PBS for two hours. Decant the solution and wash the zebrafish larva using 100 millimolar Tris HCL with a pH of 7.5 or 10 millimolar Magnesium Chloride. For de-staining, decant the solution and incubate the larva for five minutes in a series of solutions as described in the manuscript.After the final incubation, decant the solution and remove the pigment by bleaching with a solution consisting of 3%hydrogen peroxide and 0.5%potassium hydroxide. Observe once every 10 minutes until the pigment is completely removed. Rinse the zebrafish larva several times with 25%glycerin, 0.1%potassium hydroxide for 10 minutes each until there are no bubbles.Stain in the larva by soaking in one milliliter of 0.01%Alizarin. Decant the solution, and add one milliliter of 50%glycerin or 0.1%potassium hydroxide to clear the background. Store the fish in fresh 50%glycerin or 0.1%potassium hydroxide for subsequent observation.Transfer one larva at a time onto the glass slide keeping the larva in the middle of the liquid drop. Observe the larva under a stereo microscope. Turn on the camera.Open the software, and keep the default settings. Click on AE and choose an appropriate exposure time to obtain the best image. Capture all the images under the same settings.Save the images in dot tiff format for later analysis. Double click the imageJ icon. To analyze the saved images, click on file, then open, then click on image, then type and select eight-bit.Click on edit, and invert. Click on analyze, then calibrate. Select Uncalibrated OD in the pop-up interface.Check global calibration at the bottom left of the lower interface, and click okay. Click on analyze, set scale, then remove scale. Check global below, and click okay.Click on analyze, then set measurements. Select the item area in the pop-up interface. Check the limit to threshold below to measure only the selected range, and click okay.Click on image, adjust, then threshold, and slide the slider in the middle of the pop-up interface. To select the appropriate threshold so that all targets to be tested in one image are selected. And click set.Click on analyze, then measure, save the dates. Alizarin Red Staining is a sensitive and specific method for measuring changes in bone mineralization in zebrafish larvae. At nine days post fertilization, many bones of the head skeleton such as parasphenoid, opercular, ceratobranchial, and notochord are mineralized and therefore stained in red.In contrast, non-bony structures like otoliths, appear brown black. Digital analysis performed to quantify the total stained area, showed a significant decrease in the stained area for the 10 and 20 milligrams per liter of lead acetate treatment groups. The changes in bone mineralization showed dose dependency.For better staining effect and observation, it's important to remove the original pigment of zebrafish larvae, and remove the bubbles after bleaching.

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Developmental Biology

4.0K ビュー.  Medical College of Soochow University. この方法は、ゼブラフィッシュの化学物質によって誘発される骨毒性を検出するのに役立ちます。アリザリンレッド 固定組織の染色は、いくつかの標本の比較を容易にすることができる骨格変化の永久記録を生成します。この技術は、骨粗鬆症の特徴の1つが骨量の減少であるため、ゼブラフィッシュモデルで骨粗鬆症を研究するのに役立つ可能性があります。受?...