Name: Author Spotlight: Advancing Bone Cell Activity Research with Zebrafish Scales
Uploaded: 2025-01-10T12:10:00
Description: Zebrafish scales offer a variety of advantages for use in standard laboratories for teaching and research purposes. Scales are easily collected without ...

The authors thank Mount Saint Vincent University Aquatic Facility staff and all members of the Franz-Odendaal Bone Development lab for providing the necessary fish care. Specific thanks to Alisha McNeil, Keely A. MacLellan, and Shirine Jeradi for assisting in optimizing some protocols. This research was supported by funding from the Canadian Space Agency (CSA) [19HLSRM01] and the Natural Sciences and Engineering Research Council of Canada (NSERC).

All protocols described here follow the Canadian Council on Animal Care guidelines and were approved annually by the Saint Mary&#39;s University/Mount Saint Vincent University Animal Care committee under protocol numbers 20-14 and 21-12. Before carrying out this protocol, users must ensure that federal and provincial guidelines on using animals in research or teaching are followed23. The details of the reagents and equipment used in this study are listed in the Table of Materials....

Removing Zebrafish Scales for Culturing and Bone Biology Studies

<ol>
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P., Look, A. T. <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+a+model+for+the+study+of+human+cancer.">Zebrafish as a model for the study of human cancer.</a> Methods Cell Biol. 105, 309-337 (2011).</li><li>Choe, C. P., 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=Transgenic+fluorescent+zebrafish+lines+that+have+revolutionized+biomedical+research.">Transgenic fluorescent zebrafish lines that have revolutionized biomedical research.</a> Lab Anim Res. 37, 1-29 (2021).</li><li>Brito, R. S., Canedo, A., Farias, D., Rocha, T. 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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=Regenerating+zebrafish+scales+express+a+subset+of+evolutionary+conserved+genes+involved+in+human+skeletal+disease.">Regenerating zebrafish scales express a subset of evolutionary conserved genes involved in human skeletal disease.</a> BMC Biol. 20 (1), 1-25 (2022).</li><li>Sire, J., Girondot, M., Babiar, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marking+zebrafish,+Danio+rerio+(Cyprinidae),+using+scale+regeneration.">Marking zebrafish, Danio rerio (Cyprinidae), using scale regeneration.</a> J Exp Zool. 286 (3), 297-304 (2000).</li><li>Carvajal-Agudelo, J. D., McNeil, A., Franz-Odendaal, T. 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J., Fulbright, A. N., Parichy, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/&#946;-catenin+regulates+an+ancient+signaling+network+during+zebrafish+scale+development.">Wnt/&#946;-catenin regulates an ancient signaling network during zebrafish scale development.</a> Elife. 7, e37001 (2018).</li><li>Carnovali, M., Luzi, L., Banfi, G., Mariotti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+hyperglycemia+affects+bone+metabolism+in+adult+zebrafish+scale+model.">Chronic hyperglycemia affects bone metabolism in adult zebrafish scale model.</a> Endocr. 54, 808-817 (2016).</li><li>Burns, A. R., Guillemin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+scales+of+the+zebrafish:+Host-microbiota+interactions+from+proteins+to+populations.">The scales of the zebrafish: Host-microbiota interactions from proteins to populations.</a> COMICR. 38, 137-141 (2017).</li><li>Vieira, E. F. S., 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+use+of+freshwater+fish+scale+of+the+species+Leporinus+elongatus+as+adsorbent+for+anionic+dyes:+An+isothermal+calorimetric+study.">The use of freshwater fish scale of the species Leporinus elongatus as adsorbent for anionic dyes: An isothermal calorimetric study.</a> J Therm Anal Calorim. 109 (3), 1407-1412 (2012).</li><li>Ighalo, J. O., Eletta, O. A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+biosorption+of+pollutants+by+fish+scales:+A+mini-review.">Recent advances in the biosorption of pollutants by fish scales: A mini-review.</a> Chem Eng Commun. 208 (9), 1301-1312 (2021).</li><li>Eletta, O. A. A., Ighalo, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+fish+scales+as+a+source+of+biosorbent+for+the+removal+of+pollutants+from+industrial+effluents.">A review of fish scales as a source of biosorbent for the removal of pollutants from industrial effluents.</a> J Res Inf Civ Eng. 16 (1), 2479-2510 (2019).</li><li>Hill, A. J., Teraoka, H., Heideman, W., Peterson, R. E. <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+a+model+vertebrate+for+investigating+chemical+toxicity.">Zebrafish as a model vertebrate for investigating chemical toxicity.</a> Toxicol. Sci. 86 (1), 6-19 (2005).</li><li>. CCAC Canadian Council of Animal Care (CCAC) Available from: <a target="_blank" href="https://ccac.ca/">https://ccac.ca/</a> (2024)</li><li>Suzuki, N., Hattori, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melatonin+suppresses+osteoclastic+and+osteoblastic+activities+in+the+scales+of+goldfish.">Melatonin suppresses osteoclastic and osteoblastic activities in the scales of goldfish.</a> J. Pineal Res. 33 (4), 253-258 (2002).</li><li>Suzuki, 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=Effect+of+vibration+on+osteoblastic+and+osteoclastic+activities:+Analysis+of+bone+metabolism+using+goldfish+scale+as+a+model+for+bone.">Effect of vibration on osteoblastic and osteoclastic activities: Analysis of bone metabolism using goldfish scale as a model for bone.</a> ASR. 40 (11), 1711-1721 (2007).</li><li>Kawakami, 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=z+Trap:+zebrafish+gene+trap+and+enhancer+trap+database.">z Trap: zebrafish gene trap and enhancer trap database.</a> BMC Dev Biol. 10, 1-10 (2010).</li><li>. ZFIN The Zebrafish Model Organism Database. Available from: <a target="_blank" href="https://zfin.org/">https://zfin.org/</a> (2024)</li><li>Edsall, S. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+scale+collagen-A+novel+material+for+corneal+tissue+engineering.">Fish scale collagen-A novel material for corneal tissue engineering.</a> Artif Organs. 36 (9), 829-835 (2012).</li><li>Zhao, S., 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=A+novel+porous+nanocomposite+of+sulfur/carbon+obtained+from+fish+scales+for+lithium-sulfur+batteries.">A novel porous nanocomposite of sulfur/carbon obtained from fish scales for lithium-sulfur batteries.</a> J Mater Chem. 1 (10), 3334-3339 (2013).</li><li>Kumar, R., 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=A+noninvasive+technique+for+rapid+extraction+of+DNA+from+fish+scales.">A noninvasive technique for rapid extraction of DNA from fish scales.</a> IJEB. 45 (11), 992-999 (2007).</li><li>Hutchinson, J. J., Trueman, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotope+analyses+of+collagen+in+fish+scales:+Limitations+set+by+scale+architecture.">Stable isotope analyses of collagen in fish scales: Limitations set by scale architecture.</a> J Fish Biol. 69 (6), 1874-1880 (2006).</li><li>Kumari, S., Rath, P. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+morphology,+structures+and+mechanical+properties+of+teleost+fish+scales+collected+from+New+Zealand.">Comparison of the morphology, structures and mechanical properties of teleost fish scales collected from New Zealand.</a> J. Bionic Eng. 16, 328-336 (2019).</li><li>Witzmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+histological+changes+of+dermal+scales+during+the+fish-to-tetrapod+transition.">Morphological and histological changes of dermal scales during the fish-to-tetrapod transition.</a> Acta Zool. 92 (3), 281-302 (2011).</li><li>Iba&#241;ez, A. L., Cowx, I. G., O'Higgins, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+morphometric+analysis+of+fish+scales+for+identifying+genera,+species,+and+local+populations+within+the+Mugilidae.">Geometric morphometric analysis of fish scales for identifying genera, species, and local populations within the Mugilidae.</a> Can J Fish Aquat Sci. 64 (8), 1091-1100 (2007).</li></ol>

Zebrafish scales are an easily accessible in vitro model to study a variety of different biological processes that can be maintained for up to 2 days using a culturing method and an incubator to simulate their natural environment11. Scales have a regular and proportional distribution of cells present, which enables researchers to view, count, and label cells and to conduct simple cell biology experiments using basic laboratory equipment. Furthermore, the entire scale is easily visible wit...

In recent years, zebrafish (Danio rerio) have emerged as a valuable model organism in various scientific fields, including genetics, developmental biology, and toxicology1,2,3. Zebrafish are small tropical freshwater fish native to the rivers of Southeast Asia4. They have become a popular model organism in biology due to their easy maintenance, small size, rapid reproductive cycle, and translucent embryos1. This allows for easy observation of their internal development, making them ideal for studying various biological processes in developmental biology. Zebrafish share genetic similarities with humans; approximately 70% of human genes have at least one zebrafish counterpart, making them an excellent model for studying genetic disorders and diseases in humans5,6. By manipulating specific genes in zebrafish, researchers can gain valuable insights into the function and behavior of genes, which can then be applied to human health research6, gene editing, and the creation of transgenic lines7,8.
Zebrafish scales regenerate after removal9,10 and can be cultured for one to three days with a basic (non-CO2) incubator while they are used for experiments11. The use of zebrafish scales is advantageous in basic laboratories as they can be removed from anesthetized fish without the need for euthanasia. Zebrafish scales are a component of the dermal skeleton, consisting primarily of two layers: the internal layer, which is thick and made of partially mineralized tissue formed by layers of collagen fibrils, and the external layer, which is highly mineralized and contains a network of interwoven collagen fibrils12,13&#160;(Figure 1A-C). These morphological and cellular features of the scale are easily observed under a standard compound microscope. These characteristics make zebrafish scales a good model for studying cellular and molecular. For example, zebrafish scales have been used to study bone homeostasis, including bone cell activity11,12. They have also been used to understand tissue regeneration9,14,15, gene pathways and signaling14,16, metabolism17, microbiota studies18, etc. Zebrafish, similar to other teleosts, are also highly sensitive to environmental factors such as pollutants and toxins19,20,21,22. As such, fish scales are used to study exposures to toxins in fish populations. These characteristics make scales an excellent model for teaching students about the impacts of environmental factors on living organisms. Additionally, scales from transgenic zebrafish lines, such as Tg(osterix:mCherry) and Tg(runx2a:GFP), could add another level of complexity to student experiments.
In summary, by using zebrafish scales, researchers can design and conduct experiments to study various aspects of biology, from cellular mechanisms to environmental changes. Zebrafish are also a valuable resource in higher education since they can provide hands-on experiences in experimental design, data collection, and analysis. Zebrafish can also be utilized in advanced courses and research-focused programs to explore specific areas of interest. This article describes a protocol for using zebrafish scales in research and classroom learning environments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL tubes</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>37% HCl&#160;</td>
			<td>EMD</td>
			<td>HX0603-4</td>
			<td>For pH stabilization and prepration of PRS</td>
		</tr>
		<tr>
			<td>Concave slide</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Vectashield</td>
			<td>H-1200</td>
			<td>For cell nuclei visualization</td>
		</tr>
		<tr>
			<td>Diazonium salt (Fast Blue B)</td>
			<td>Sigma</td>
			<td>D9805</td>
			<td>For AP substrate solution</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) powder&#160;</td>
			<td>Sigma&#160;</td>
			<td>D5523</td>
			<td>For scale culture media&#160;</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate (MS222)&#160;</td>
			<td>Sigma&#160;</td>
			<td>E10521</td>
			<td>For preparation of 0.1% MS222</td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>Sigma&#160;</td>
			<td>F4135</td>
			<td>For scale culture media&#160;</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid&#160;</td>
			<td>Fisher&#160;</td>
			<td>A38 212</td>
			<td>For acetate buffer</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR</td>
			<td>BDH1172-4LP</td>
			<td>For 80% glycerol - storage scales</td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>ThermoFisher&#160;</td>
			<td>15630106</td>
			<td>For scale culture media&#160;</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl&#160;</td>
			<td>Sigma</td>
			<td>P217-10</td>
			<td>For preparation of PBS</td>
		</tr>
		<tr>
			<td>Lysotracker</td>
			<td>ThermoFisher</td>
			<td>L7528</td>
			<td>For lysosomes&#160; visualization</td>
		</tr>
		<tr>
			<td>Maleic acid</td>
			<td>Sigma</td>
			<td>M0375</td>
			<td>For Tris-Maleate buffer&#160;</td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide</td>
			<td>Sigma</td>
			<td>319937</td>
			<td>For AP substrate solution</td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide&#160;</td>
			<td>Sigma</td>
			<td>319937</td>
			<td>For Enzyme Substrate Solution</td>
		</tr>
		<tr>
			<td>Na2HPO4&#160;</td>
			<td>EMD</td>
			<td>SX0720-1</td>
			<td>For preparation of PBS</td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Sigma&#160;</td>
			<td>S9888</td>
			<td>For preparation of PBS</td>
		</tr>
		<tr>
			<td>NaNO2</td>
			<td>Sigma</td>
			<td>S-2252</td>
			<td>For 4% NaNO2</td>
		</tr>
		<tr>
			<td>NaOH&#160;</td>
			<td>Sigma</td>
			<td>S5881</td>
			<td>For preparation of 4% PFA</td>
		</tr>
		<tr>
			<td>NaOH&#160;</td>
			<td>Sigma&#160;</td>
			<td>S5881</td>
			<td>For scale fixation and pH stabilization</td>
		</tr>
		<tr>
			<td>Napthol-AS-TR-phosphate</td>
			<td>Sigma</td>
			<td>N6125</td>
			<td>For AP substrate solution</td>
		</tr>
		<tr>
			<td>Napthol-AS-TR-phosphate&#160;</td>
			<td>Sigma</td>
			<td>N6125</td>
			<td>For Enzyme Substrate Solution</td>
		</tr>
		<tr>
			<td>Pararosaniline hydrochloride&#160;</td>
			<td>Sigma</td>
			<td>P3750</td>
			<td>For PRS</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin 5000 units penicillin and 5mg streptomyocin/ml</td>
			<td>ThermoFisher&#160;</td>
			<td>15140122</td>
			<td>For scale culture media&#160;</td>
		</tr>
		<tr>
			<td>Personal protective equipment (PPE): disposable nitrile gloves, safety glasses/splash goggles and lab coat.</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA&#160;</td>
			<td>Sigma&#160;</td>
			<td>P6148</td>
			<td>For scale fixation</td>
		</tr>
		<tr>
			<td>Sodium acetate&#160;</td>
			<td>Sigma&#160;</td>
			<td>S2889</td>
			<td>For acetate buffer</td>
		</tr>
		<tr>
			<td>Standard compound microscope</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir plate</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Tartaric acid</td>
			<td>Sigma</td>
			<td>T6521</td>
			<td>For Tartrate buffer</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Roche</td>
			<td>03 118 142 001</td>
			<td>For Tris-Maleate buffer&#160;</td>
		</tr>
	</tbody>
</table>

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 

In Vitro Culturing Technique for Studying Cellular Dynamics in Zebrafish Scales

Our research explores the effects of environmental disturbances of bone tissue. We are specifically interested in understanding the effects of simulated microgravity and vibration on adults and developing bone. We use zebrafish as a model organism of choice because it offers many advantages for biological research.Our protocol offers advantages over previous protocols because it limits the need for a carbon dioxide incubator and it uses standard laboratory equipment. Our lab is trying to unravel the communication dynamics between different types of bone cells when they are subjected to an environmental disturbance, such as gravity or vibration. In the future, we would like to be able to mitigate the negative effects of space travel on bone tissue.

Recently, this scale culturing protocol was used to study bone homeostasis11. Scales can survive in the culture medium for at least 2 days. The numbers of apoptotic cells were analyzed on each day of culture, up to a maximum of three days, by counting the labeled cells. These results showed that apoptotic cells increased significantly on the third day of culturing, indicating that culture time should be limited to two days using the method described here.
Comparing scal...

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 ...

in-vitro-culturing-technique-for-studying-cellular-dynamics-zebrafish

Research

JoVE Journal

Developmental Biology

Zebrafish Scale Collection for Bone Homeostasis Studies

To begin, dissolve 10 grams of DMEM powder and 5.95 grams of HEPES in one liter of distilled water, and adjust the pH of the media to 6.91. Aliquot the solution into smaller containers and store them at four degrees Celsius. Then mix 88%DMEM medium with HEPES, 10%fetal bovine serum, and 2%penicillin streptomycin.To prepare the zebra fish, set up a container or a tank with approximately two liters of water for four to five adult zebra fish. Using a fish net, transfer four to five adult zebra fish into the prepared tank. Prepare separate containers with water for anesthetizing and recovering the zebra fish.After anesthetizing an adult zebra fish, use a plastic spoon to transfer the fish to a Petri dish lined with a wet paper towel, and place the dish under a dissecting stereomicroscope. Next, using fine forceps, gently tug on the anterior to the posterior direction to remove the scales. Place each individual scale into a separate 0.2 milliliter tube containing the scale culture medium working solution, and put the tubes inside an incubator set to 28 degrees Celsius.After scale removal, carefully return the zebra fish to the recovery tank and monitor it. Once the zebra fish has resumed movement, return it to the regular rearing tank.