Name: using alizarin red staining to detect chemically induced bone loss in zebrafish larvae
Uploaded: 2021-12-28T14:00:00
Description: Watch this Scientific Journal Video about Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae at JoVE.com

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>
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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 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 ...

Deze methode kan helpen bij het detecteren van de bottoxiciteit veroorzaakt door chemicaliën in zebravissen. Alizarine Rode kleuring van vast weefsel genereert een permanent record van skeletveranderingen die vergelijkingen van verschillende exemplaren kunnen vergemakkelijken. Deze techniek kan nuttig zijn om osteoporose in het zebravismodel te bestuderen, omdat een van de kenmerken van osteoporose de afname van de botmassa is.Verwijder 10 zebravislarven willekeurig uit elke groep negen dagen na de bevruchting en fixeer de vis in één milliliter van 2% paraform aldehyde en 1X PBS gedurende twee uur. Decanteer de oplossing en was de zebravislarve met 100 millimol Tris HCL met een pH van 7,5 of 10 millimol magnesiumchloride. Voor het ontkleuren decanteert u de oplossing en incubeert u de larve gedurende vijf minuten in een reeks oplossingen zoals beschreven in het manuscript.Na de laatste incubatie decanteert u de oplossing en verwijdert u het pigment door te bleken met een oplossing bestaande uit 3% waterstofperoxide en 0,5% kaliumhydroxide. Observeer eens in de 10 minuten totdat het pigment volledig is verwijderd. Spoel de zebravislarve meerdere keren met 25% glycerine, 0,1% kaliumhydroxide gedurende 10 minuten elk totdat er geen bubbels zijn.Kleur in de larve door te weken in een milliliter van 0,01% Alizarine. Decanteer de oplossing en voeg een milliliter van 50% glycerine of 0,1% kaliumhydroxide toe om de achtergrond te verwijderen. Bewaar de vis in verse 50% glycerine of 0,1% kaliumhydroxide voor latere observatie.Breng één larve per keer over op de glasglaas en houd de larve in het midden van de vloeistofdruppel. Observeer de larve onder een stereomicroscoop. Schakel de camera in.Open de software en behoud de standaardinstellingen. Klik op AE en kies een geschikte belichtingstijd om de beste afbeelding te verkrijgen. Leg alle afbeeldingen vast onder dezelfde instellingen.Sla de afbeeldingen op in dot tiff-indeling voor latere analyse. Dubbelklik op het imageJ-pictogram. Om de opgeslagen afbeeldingen te analyseren, klikt u op bestand, vervolgens op openen, klikt u vervolgens op afbeelding en typt u en selecteert u acht-bits.Klik op bewerken en keer om. Klik op analyseren en vervolgens kalibreren. Selecteer Niet-gekalibreerde OD in de pop-upinterface.Controleer de algemene kalibratie linksonder in de onderste interface en klik op OK. Klik op analyseren, stel schaal in en verwijder vervolgens schaal. Controleer hieronder globaal en klik op OK.Klik op analyseren en stel vervolgens metingen in. Selecteer het itemgebied in de pop-upinterface. Controleer de limiet tot de drempel hieronder om alleen het geselecteerde bereik te meten en klik op OK.Klik op afbeelding, pas aan, drempel en schuif de schuifregelaar in het midden van de pop-upinterface. De juiste drempelwaarde selecteren zodat alle te testen doelen in één afbeelding worden geselecteerd. En klik op instellen.Klik op analyseren, meet vervolgens, sla de datums op. Alizarine Red Staining is een gevoelige en specifieke methode voor het meten van veranderingen in botmineralisatie bij zebravislarven. Negen dagen na de bevruchting zijn veel botten van het hoofdskelet zoals parasphenoïde, operculair, ceratobranchiaal en notochord gemineraliseerd en daarom rood gekleurd.Daarentegen lijken niet-benige structuren zoals otolieten bruinzwart. Digitale analyse uitgevoerd om het totale gebeitste gebied te kwantificeren, toonde een significante afname van het gekleurde gebied voor de 10 en 20 milligram per liter loodacetaatbehandelingsgroepen. De veranderingen in botmineralisatie toonden dosisafhankelijkheid.Voor een beter kleuringseffect en observatie is het belangrijk om het oorspronkelijke pigment van zebravislarven te verwijderen en de bubbels na het bleken te verwijderen.

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