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.
1. Preparation of reagents for scale culture
<ol>
	<li>Prepare DMEM medium with HEPES (10 g of DMEM powder, 5.95 g of HEPES, in 1 L of distilled water or dH2O, pH: 6.91), aliquot, and store at 4 &#176;C.</li>
	<li>Prepare the culture medium - working solution (88% DMEM medium with HEPES, 10% fetal bovine serum, and 2% penicillin-streptomycin [5000 units penicillin and 5 mg streptomycin per mL] adjusted to the volume needed on the day the experiment is performed. 
	NOTE: The working solution must be prepared fresh daily and cannot be stored. The culture medium must be kept cold (at 4 &#176;C or on ice) until used.</li>
	<li>Aliquot the culture medium working solution into the containers in which the experiment will be carried out. In this case, 0.2 mL tubes or a 96-well plate were used. Maintain these aliquots on ice until required. 
	NOTE: This solution should be prepared fresh and cannot be stored for more than 1 day. Refer to Table 1 for all reagents.</li>
</ol>
2. Removing scales from live fish
<ol>
	<li>Prepare a container or tank for the zebrafish with a water volume of approximately 2 L for 4-5 adult fish. 
	NOTE: The typical parameters of the water that are appropriate for zebrafish are pH 7.5, temperature 28.5 &#176;C, conductivity 640-781 &#181;S, and a 12-12-h dark-light cycle. Ensure that temperature is constantly maintained in the tank.</li>
	<li>Use a fish net to capture 4-5 adult zebrafish and place them in the zebrafish holding tank prepared above.</li>
	<li>Prepare a container with at least 60 mL of water where the fish will be anesthetized. 
	NOTE: The fish must be able to move in the container and be deep enough to prevent them from jumping out. This container should contain buffered 0.01% tricaine methane-sulfonate (MS222), which should be made up of zebrafish water (Dilute 0.1% MS222, which is prepared with 0.3 g of MS222, 615 &#181;L of 1 M NaOH in 300 mL of zebrafish water system, ten times). 
	CAUTION: Individuals conducting these experiments must have the institutional animal ethics board&#39;s approval to catch, anesthetize, and handle zebrafish. Similarly, the anesthetic used is a low concentration of tricaine methane-sulfonate (MS222), which is a chemical commonly used in fish biology but one that requires proper handling, such as appropriate personal protective equipment, including disposable nitrile gloves, safety glasses/splash goggles and lab coat (Refer to the safety data sheet of MS222 for more information).</li>
	<li>Prepare a recovery container with the same specifications as used previously in step 1 (at least 2 L of water) where the fish will be placed after scale removal. This container should have enough space and the correct water parameters, as noted above.</li>
	<li>Using a net, carefully transfer a single adult zebrafish to the container with 0.01% MS222. Wait until the fish is completely immobile and has minimal opercular movement (this usually takes less than 10 min). 
	NOTE: This entire step is carried out one fish at a time. Do not anesthetize the next fish until the scales have been successfully removed from the fish that is being handled and until that fish has been transferred to the recovery container.</li>
	<li>Individually place the adult zebrafish on an inverted Petri dish lined with a wet paper towel.</li>
	<li>Under a dissecting stereomicroscope and using fine forceps, remove a maximum of 10-12 scales from an individual fish. The scales are removed from the lateral region of the fish, where the scales are bigger and have a more constant size compared with the other parts of the body (Figure 2). Both sides of the fish can be used.
	<ol>
		<li>Remove the scales one at a time by gently tugging in the natural direction of the scale (i.e., in the direction from anterior to posterior). This should be done as quickly as possible.</li>
	</ol>
	</li>
	<li>Place each individual scale in a separate 0.2 mL tube containing the scale culture medium-working solution (prepared in step 1.1) to prevent the scales from degradation. A 96-well plate can be used as an alternative. 
	NOTE: Isolation of individual scales in separate tubes or wells prevents the scales from sticking together and allows the tracking of individual scales that may be needed for multiple experiments.</li>
	<li>If the specific experiment requires a scale treatment, apply it in this step. For examples of some protocols, refer to steps 4 and 5.</li>
	<li>Put the tubes with the scales inside an incubator at 28 &#176;C. The culture medium should be changed every 24 h (with the culture medium-working solution). A non-CO2 incubator was used for the experiments outlined here.</li>
	<li>After scale removal, carefully return the zebrafish to the recovery tank and monitor until movement resumes. A pipette or aeration stone is used to provide air in the tank to speed up recovery.</li>
	<li>Once movement is restored, return the zebrafish to a regular rearing tank. Remember to keep the fish that were used separately from any other fish so that they can recover separately and to keep track of which fish are actively regenerating their scales.</li>
	<li>Wait ~2 weeks before repeating scale removal from these same fish. This allows time for them to regenerate their scales naturally.</li>
</ol>
3. Fixation of scales
<ol>
	<li>Wash the scales three times with 200 &#181;L of 1x of phosphate-buffered saline (PBS) prepared from 10x PBS (for 1 L, add 80 g of NaCl, 2 g of KCl, and 11.2 g of Na2HPO4, add dH2O until 1 L).</li>
	<li>Remove the 1x PBS and add 200 &#181;L of 4% paraformaldehyde (PFA) overnight at 4 &#176;C. For 100 ml of PFA, add 4 g of paraformaldehyde and 0.1 g of NaOH, then add 1x PBS until 100 mL. Mix on a hot plate set to low heat and stir until the solution is clear, then allow the solution to cool down and stabilize the pH to 7.4. Store at -20 &#176;C. For details, please see Supplementary File 1 (Table 1).</li>
	<li>After fixation, wash the scales three times with 200 &#181;L of 1x PBS. Store the scales at 4 &#176;C. 
	CAUTION: PFA is harmful. Refer to the safety data sheet for more information. All these processes require proper handling, such as appropriate personal protective equipment, including disposable nitrile gloves, safety glasses/splash goggles, and a lab coat.</li>
</ol>
4. Bone homeostasis
NOTE: Two bone homeostasis procedures are described below. Tartrate-resistant acid phosphatase (TRAP) and alkaline phosphate (AP) staining are typically used to identify active osteoclasts and osteoblasts, respectively. Osteoclasts resorb bone matrix while osteoblasts deposit bone matrix. AP is the main enzyme released by osteoblasts during bone formation, while TRAP is secreted by osteoclasts into the acidic space between the osteoclast and bone matrix, assisting in bone breakdown and resorption24,25,26. This protocol is also available on The Zebrafish Information Network27 and was previously published28. Alkaline phosphatase is an enzyme produced by multiple cell types as part of the metabolic process. This process is not wholly specific to osteoblasts, but in bone tissue, it is used as a marker for osteoblast function.
<ol>
	<li>Tartrate-resistant acid phosphatase (TRAP) staining
	<ol>
		<li>Place each fixed scale into a 0.2 mL tube or a 96-well plate with 200 &#181;L of dH2O. Wash the scales by adding and removing around 150 &#181;L of dH2O for 15 min three times. For details, please see Supplementary File 1 (Table 2).</li>
		<li>Remove the scales from the 0.2 mL tube or well and place them in a new tube that contains 200 &#181;L of 50 mM tartrate buffer. Incubate for 1 h at room temperature.
		<ol>
			<li>For 100 mM of tartrate buffer, prepare a glass container, add 2.3 g of tartaric acid, and mix it with 0.2 M acetate buffer (pH 5.5) until 100 mL of solution. Stir well and store at 4 &#176;C. To prepare 50 mM of tartrate buffer, add equal volumes of dH2O and 100 mM tartrate buffer. For details, please see Supplementary File 1 (Table 3 and Table 4).</li>
		</ol>
		</li>
		<li>Remove the tartrate buffer from the tube and add 200 &#181;L of TRAP substrate solution. Incubate the samples for 1 h at room temperature. Ensure samples remain in the dark.
		<ol>
			<li>Prepare a dark glass container and add 30 mL of 100 mM tartrate buffer, 1 mL of hexazotized PRS (Pararosaniline hydrochloride solution), and 2 mL of enzyme substrate solution. Stir well. 
			NOTE: This solution cannot be stored; keep it in the dark at room temperature while using. For the solutions needed to prepare the TRAP substrate solution, please see Supplementary File 1 (Tables 5-9).</li>
		</ol>
		</li>
		<li>Remove the TRAP substrate solution and wash the scales by adding and removing around 150 &#181;L of dH2O for 15 min, three times.</li>
		<li>Remove the scales from the tube and place them into a new tube or well with 200 &#181;L of 80% glycerol (for 100 mL, mix 80 mL of glycerol with 20 mL of dH2O) wrapped in aluminum foil to avoid fading and store them at 4 &#176;C.</li>
		<li>To visualize the scales under the microscope, use a depression or concave slide.</li>
	</ol>
	</li>
	<li>Alkaline phosphate (AP) staining
	<ol>
		<li>Place each fixed scale in a 0.2 mL tube or 96-well plate with 200 &#181;L of dH2O and wash them by adding and removing around 150 &#181;L of dH2O for 15 min, three times. For details, please see Supplementary File 1 (Table 2).</li>
		<li>Remove the scales from the 0.2 mL tubes or wells and place them into a new tube that contains 200 &#181;L of tris-maleate buffer and incubate it for 1 h at room temperature.
		<ol>
			<li>For this buffer, prepare a glass container and add 0.605 g of tris buffer and 0.55 g of maleic acid. Add dH2O until 25 mL and stir well. Stabilize to pH 8.3. Store at 4 &#176;C. For details, please see Supplementary File 1 (Table 10).</li>
		</ol>
		</li>
		<li>Remove the tris-maleate buffer from the tube and add 200 &#181;L of AP substrate solution. Incubate it for 1 h at room temperature. Ensure samples remain in the dark.
		<ol>
			<li>For this solution, prepare a dark glass container, add 8 mg of diazonium salt, and mix it with 0.1 mL of napthol-AS-TR-phosphate in N,N-dimethylformamide, and 10 mL of tris-maleate buffer (pH 8.3), stir well. Prepare this solution fresh. For details, please see Supplementary File 1 (Table 11).</li>
		</ol>
		</li>
		<li>Remove the AP substrate solution and wash the scales by adding and removing 200 &#181;L of dH2O for 15 min, three times.</li>
		<li>Remove the scales from the tube and place them into a new tube or well with 200 &#181;L of 80% glycerol wrapped in aluminum foil to avoid fading. Store at 4 &#176;C.</li>
		<li>To visualize the scales using a microscope, place the scales on a concave or depression slide. 
		CAUTION: Most of the reagents used in the TRAP and AP staining can be harmful. Refer to the safety data sheet for each chemical. All these procedures require proper handling, such as appropriate personal protective equipment, including disposable nitrile gloves, safety glasses/splash goggles, and a lab coat.</li>
	</ol>
	</li>
</ol>
5. Visualizing cell dynamics in zebrafish scales
NOTE: A range of staining methods can be used to visualize differences between scales and to label cells within the scale (Figure 3B). For example, methods to stain nuclei and lysosomes within the cells of the scale are outlined below.
<ol>
	<li>DAPI staining for cell nuclei visualization 
	NOTE: DAPI is used to mark the nuclei of all cells by labeling the DNA. DAPI is a fluorescent marker that binds strongly to regions enriched for adenine and thymine in DNA sequences.
	<ol>
		<li>Before using DAPI, ensure that scales are fixed in 4% paraformaldehyde (step 3). Wash the scales with 200 &#181;L 1x PBS for 5 min after fixing.</li>
		<li>Prepare a DAPI working solution in a 1:10 dilution as follows. Mix 1 &#181;L of DAPI with 9 &#181;L of 1x PBS to make a total volume of 10 &#181;L.</li>
		<li>After preparing the DAPI solution, use forceps or a plastic pipette to put the scale on a concave slide, remove excess PBS, and add around 10 &#181;L of the prepared DAPI solution. 
		NOTE: DAPI will stain the scale immediately, so it can be visualized using a fluorescence microscope with an excitation wavelength of 352-402 nm and an emission wavelength of 417-477 nm. DAPI will stain all cell nuclei a bright blue color. In order to see the entire scale in the field of view, the 4x objective was used. Single stained nuclei are visible using the 20x objective.</li>
		<li>Count the number of DAPI-stained nuclei in order to determine the number of cells in the scale.</li>
	</ol>
	</li>
	<li>Apoptotic staining for cell death visualization in live samples 
	NOTE: Fluorescent stains can be used to label apoptotic cells. For example, the fluorescence marker protocol used here is for acidic environments and can be used for labeling and tracing acidic organelles like lysosomes. An increase in the number of these organelles is correlated with increased cell death. This stain should be applied to unfixed scales that are either freshly pulled or have been cultured for up to 2 days.
	<ol>
		<li>Prepare a solution of the fluorescent stain for apoptosis tracking according to the manufacturer&#39;s instructions by using the culture medium as the dilutant. 
		NOTE: The concentration of the fluorescent stain can change drastically depending on the apoptosis method used. Refer to the commercial protocol for the stain of choice for specific details on recommended concentrations.</li>
		<li>Carefully remove the culture medium from each 0.2 mL tube or well from a 96-well plate if the scales were previously cultured.</li>
		<li>Add enough fluorescent staining solution to cover the whole scale (around 50 &#181;L).</li>
		<li>Cover all the tubes with aluminum foil to avoid fading and leave for 30 min.</li>
		<li>Remove the fluorescent staining solution from the tubes or wells.</li>
		<li>Wash samples by adding 200 &#181;L of 1x PBS to the tubes or wells for 5 min.</li>
		<li>Remove the PBS and add 200 &#181;L of 4% PFA to the tubes or wells to fix the samples for 1 h at room temperature.</li>
		<li>Wash samples by adding 200 &#181;L 1x PBS to the tubes or wells for 5 min.</li>
		<li>Transfer the scales into a new 0.2 mL tube or well, and add 200 &#181;L of fresh 1x PBS. Cover these tubes/wells in aluminum foil to avoid fading. Store at 4 &#176;C.</li>
		<li>To visualize the scales with a fluorescence microscope, use a concave or depression slide, and the correct filter. The fluorescent signal used here has an excitation and emission maximum of 577-590 nm. The fluorescent stain shows apoptotic cells containing lysosomes in red.</li>
		<li>Count the number of stained cells in order to determine the number of cells undergoing apoptosis. 
		NOTE: Aluminum foil is used during staining and storage to avoid fading. Samples can be stored in the dark for a couple of hours or even days, depending on which fluorescent stain is used. To store fluorescent samples for longer periods, use a fixation and mounting protocol that is compatible with the fluorescent stain. All fluorescent staining was visualized using a fluorescence microscope with at least 20x magnification. The filter used was as follows: for DAPI 417-477 nm; for apoptosis tracking 577-590 nm.</li>
	</ol>
	</li>
</ol>

Removing Zebrafish Scales for Culturing and Bone Biology Studies

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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. C., Franz-Odendaal, T. 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+quick+whole-mount+staining+protocol+for+bone+deposition+and+resorption.">A quick whole-mount staining protocol for bone deposition and resorption.</a> Zebrafish. 7 (3), 275-280 (2010).</li><li>Howe, D. G., 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+Model+Organism+Database:+Increased+support+for+mutants+and+transgenics.">the Zebrafish Model Organism Database: Increased support for mutants and transgenics.</a> Nucleic Acids Res. 41 (D1), D854-D860 (2012).</li><li>Singleman, C., Holtzman, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+maturation+in+the+zebrafish,+Danio+rerio:+A+staging+tool+for+teaching+and+research.">Growth and maturation in the zebrafish, Danio rerio: A staging tool for teaching and research.</a> Zebrafish. 11 (4), 396-406 (2014).</li><li>Barresi, M. 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=Zebrafish+in+the+Classroom.">Zebrafish in the Classroom.</a> Zebrafish. 5 (3), 205-208 (2008).</li><li>Ghods, 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=On+the+regeneration+of+fish+scales:+Structure+and+mechanical+behavior.">On the regeneration of fish scales: Structure and mechanical behavior.</a> J Exp Biol. 223 (10), jeb211144 (2020).</li><li>Krishnan, S., Sekar, S., Katheem, M. F., Krishnakumar, S., Sastry, T. 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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+characterization+of+chitin+and+chitosan+from+(Labeo+rohit)+fish+scales.">Extraction and characterization of chitin and chitosan from (Labeo rohit) fish scales.</a> Procedia Mater Sci. 6, 482-489 (2014).</li><li>Stepnowski, P., &#211;lafsson, G., Helgason, H., Jastorff, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+study+on+chemical+and+physical+principles+of+astaxanthin+sorption+to+fish+scales+towards+applicability+in+fisheries+waste+management.">Preliminary study on chemical and physical principles of astaxanthin sorption to fish scales towards applicability in fisheries waste management.</a> Aquac. 232 (1-4), 293-303 (2004).</li><li>Ghods, S., Murcia, S., Ossa, E. A., Arola, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designed+for+resistance+to+puncture:+The+dynamic+response+of+fish+scales.">Designed for resistance to puncture: The dynamic response of fish scales.</a> J Mech Behav Biomed Mater. 90, 451-459 (2019).</li><li>Yang, W., 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=Protective+role+of+Arapaima+gigas+fish+scales:+Structure+and+mechanical+behavior.">Protective role of Arapaima gigas fish scales: Structure and mechanical behavior.</a> Acta Biomater. 10 (8), 3599-3614 (2014).</li><li>Zhu, D., Zhang, C., Liu, P., Jawad, L. 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>

The authors declare that they have no conflict of interest.

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.

1.2K Views.  Mount Saint Vincent University.  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.

Removing Zebrafish Scales for Culturing and Bone Biology Studies

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