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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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 ...

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