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>
	<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 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.

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

Research

JoVE Journal

Developmental Biology

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

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 ...

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 ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts

Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. 8-hydroxy-2&#39;deoxygenase (8-OHdG) is a marker of oxidative DNA damage and &#947;H2AX is a sensitive marker for DNA double strand breaks. In this study, we aimed to detect PM2.5-induced 8-OHdG and &#947;H2AX changes in the heart of zebrafish embryos using an immunofluorescence assay. Zebrafish embryos were treated with extractable organic matters (EOM) from PM2.5 at 5 &#956;g/mL in the presence or absence of antioxidant N-acetyl-L-cysteine (NAC, 0.25 &#956;M) at 2 h post fertilization (hpf). DMSO was used as a vehicle control. At 72 hpf, hearts were dissected from embryos using a syringe needle and fixed and permeabilized. After being blocked, samples were probed with primary antibodies against 8-OHdG and &#947;H2AX. Samples were then washed and incubated with secondary antibodies. The resulting images were observed under fluorescence microscopy and quantified using ImageJ. The results show that EOM from PM2.5 significantly enhanced 8-OHdG and &#947;H2AX signals in the heart of zebrafish embryos. However, NAC, acting as a reactive oxygen species (ROS) scavenger, partially counteracted the EOM-induced DNA damage. Here, we present an immunofluorescence protocol for investigating the role of DNA damage in PM2.5-induced heart defects that can be applied to the detection of environmental chemical-induced protein expression changes in the hearts of zebrafish embryos.

Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts

This protocol uses an immunofluorescence assay to detect PM2.5-induced DNA damage in the dissected hearts of zebrafish embryos.

Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. ...

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control ...

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at 2 days post fertilization. Consisting of only two nephrons, the pronephros serves as the sole kidney during larval life until more renal function is required due to the increasing body mass. To cope with this higher demand, the mesonephros (adult kidney) begins to form during metamorphosis. The new primary nephrons fuse to the pronephros and form connected lumens. Then, secondary nephrons fuse to primary ones (and so on) to create a branching network in the mesonephros. The vast majority of research is focused on the pronephros due to the ease of using embryos. Thus, there is a need to develop techniques to study older and larger larvae and juvenile fish to better understand mesonephros development. Here, an in situ hybridization protocol for gene expression analysis is optimized for probe penetration, washing of probes and antibodies, and bleaching of pigments to better visualize the mesonephros. The Tg(lhx1a-EGFP) transgenic line is used to label progenitor cells and the distal tubules of nascent nephrons. This protocol fills a gap in mesonephros research. It is a crucial model for understanding how new kidney tissues form and integrate with existing nephrons and provide insights into regenerative therapies.

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish is an important model for understanding kidney development. Here, an in situ hybridization protocol is optimized to detect gene expression in zebrafish larvae and juveniles during mesonephros development.

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...

Processing Gut Cells from Zebrafish Larvae for Single-Cell RNA Sequencing

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

Author Spotlight: Isolating and Analyzing Intestinal Cells of Zebrafish Larvae for Investigating Transcriptomic Aspects of Gastrointestinal Development 

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.