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.

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...

Author Spotlight: Advancing Bone Cell Activity Research with Zebrafish Scales 

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.

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 ...

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

Research

JoVE Journal

Developmental Biology

2.5K 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.

Video: Author Spotlight: Advancing Bone Cell Activity Research with Zebrafish Scales 

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 ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis has become a central readout of endothelial progenitor cell functionality, and therefore, several attempts have been made to improve both in vitro and in vivo vasculogenesis models. However, standard methods are limited in scope, with static measurements failing to capture many aspects of this highly dynamic process. Therefore, the goal of developing this novel protocol was to assess the kinetics of in vitro vasculogenesis in order to quantitate rates of network formation and stabilization, as well as provide insight into potential mechanisms underlying vascular dysfunction. Application of this protocol is demonstrated using fetal endothelial colony forming cells (ECFCs) exposed to maternal diabetes mellitus. Fetal ECFCs were derived from umbilical cord blood following birth, cultured, and plated in slides containing basement membrane matrix, where they underwent vasculogenesis. Images of the entire slide wells were acquired using time-lapse phase contrast microscopy over 15 hours. Images were analyzed for derivation of quantitative data using an analysis software called Kinetic Analysis of Vasculogenesis (KAV). KAV uses image segmentation followed by skeletonization to analyze network components from stacks of multi-time point phase contrast images to derive ten parameters (9 measured, 1 calculated) of network structure including: closed networks, network areas, nodes, branches, total branch length, average branch length, triple-branched nodes, quad-branched nodes, network structures, and the branch to node ratio. Application of this protocol identified altered rates of vasculogenesis in ECFCs obtained from pregnancies complicated by diabetes mellitus. However, this technique has broad implications beyond the scope reported here. Implementation of this approach will enhance mechanistic assessment and improve functional readouts of vasculogenesis and other biologically important branching processes in numerous cell types or disease states.

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Here, we present a protocol for time-lapse imaging and analysis of vasculogenesis in vitro using phase contrast microscopy coupled with the open source software, Kinetic Analysis of Vasculogenesis. This protocol can be applied to quantitatively assess the vasculogenic potential of numerous cell types or experimental conditions that model vascular disease.

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...