<meta charset="utf8"></head><body>Rimozione delle squame di pesce zebra per studi di coltura e biologia ossea

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+scale+collagen-A+novel+material+for+corneal+tissue+engineering.">Fish scale collagen-A novel material for corneal tissue engineering.</a> Artif Organs. 36 (9), 829-835 (2012).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+porous+nanocomposite+of+sulfur/carbon+obtained+from+fish+scales+for+lithium-sulfur+batteries.">A novel porous nanocomposite of sulfur/carbon obtained from fish scales for lithium-sulfur batteries.</a> J Mater Chem. 1 (10), 3334-3339 (2013).</li><li>Kumar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+noninvasive+technique+for+rapid+extraction+of+DNA+from+fish+scales.">A noninvasive technique for rapid extraction of DNA from fish scales.</a> IJEB. 45 (11), 992-999 (2007).</li><li>Hutchinson, J. J., Trueman, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotope+analyses+of+collagen+in+fish+scales:+Limitations+set+by+scale+architecture.">Stable isotope analyses of collagen in fish scales: Limitations set by scale architecture.</a> J Fish Biol. 69 (6), 1874-1880 (2006).</li><li>Kumari, S., Rath, P. 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<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

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.

<meta charset="utf8"></head><body>Autore in primo piano: Avanzamento della ricerca sull'attività delle cellule ossee con le squame di pesce zebra

Tecnica di coltura in vitro per lo studio della dinamica cellulare nelle squame di zebrafish

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.

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

In Vitro Culturing Technique for Studying Cellular Dynamics in Zebrafish Scales

1.0K Views.  Mount Saint Vincent University. La nostra ricerca esplora gli effetti dei disturbi ambientali del tessuto osseo. Siamo specificamente interessati a comprendere gli effetti della microgravità e delle vibrazioni simulate sugli adulti e sullo sviluppo osseo. Utilizziamo il pesce zebra come organismo modello di scelta perché offre molti vantaggi per la ricerca biologica.Il nostro protocollo offre vantaggi rispetto ai protocolli precedenti perché limita la necessità di un incubatore a...

Video: Autore in primo piano: Avanzamento della ricerca sull'attività delle cellule ossee con le squame di pesce zebra 

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.

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.

Ricerca

Biologia dello sviluppo

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.