We acknowledge the funding support from the Council for Scientific and Industrial Research vide project MLP2008 and the Department of Science and Technology for the project GAP165 for supporting the work presented in this manuscript. We thank Jeyashri Rengaraju and Chetan Mishra for their help with experiments.

Zebrafish experiments were performed in strict accordance with the institutional animal ethics approval (IAEC) of the CSIR-Institute of Genomics and Integrative Biology (IGIB), India (Proposal No 45a). All efforts were made to minimize animal suffering.
1. Injecting morpholino into zebrafish embryos
<ol>
	<li>Using a standard needle puller, draw very sharp and closed-tipped pipets.</li>
	<li>Load the solution containing morpholino into the micropipettes using a microloader t...

<ol>
	<li>Kelsh, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+evolution+of+pigment+patterns+in+fish.">Genetics and evolution of pigment patterns in fish.</a> Pigment Cell Research. 17 (4), 326-336 (2004).</li><li>Jablonski, 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=The+evolution+of+human+skin+and+skin+color.">The evolution of human skin and skin color.</a> Annual Review of Anthropology. 33, 585-623 (2004).</li><li>Hoekstra, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics,+development+and+evolution+of+adaptive+pigmentation+in+vertebrates.">Genetics, development and evolution of adaptive pigmentation in vertebrates.</a> Heredity. 97 (3), 222-234 (2006).</li><li>Quigley, I. K., Parichy, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pigment+pattern+formation+in+zebrafish:+a+model+for+developmental+genetics+and+the+evolution+of+form.">Pigment pattern formation in zebrafish: a model for developmental genetics and the evolution of form.</a> Microscopy Research and Technique. 58 (6), 442-455 (2002).</li><li>Meyers, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+development+of+a+vertebrate+model+organism.">Zebrafish: development of a vertebrate model organism.</a> Current Protocols Essential Laboratory Techniques. 16 (1), 19 (2018).</li><li>Kelsh, R. N., Schmid, B., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+melanophore+development+in+zebrafish+embryos.">Genetic analysis of melanophore development in zebrafish embryos.</a> Developmental Biology. 225 (2), 277-293 (2000).</li><li>Rocha, M., Singh, N., Ahsan, K., Beiriger, A., Prince, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+development:+insights+from+the+zebrafish.">Neural crest development: insights from the zebrafish.</a> Developmental Dynamics. 249 (1), 88-111 (2020).</li><li>Carney, T. 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=A+direct+role+for+Sox10+in+specification+of+neural+crest-derived+sensory+neurons.">A direct role for Sox10 in specification of neural crest-derived sensory neurons.</a> Development. 133 (23), 4619-4630 (2006).</li><li>Curran, K., Raible, D. W., Lister, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxd3+controls+melanophore+specification+in+the+zebrafish+neural+crest+by+regulation+of+Mitf.">Foxd3 controls melanophore specification in the zebrafish neural crest by regulation of Mitf.</a> Developmental Biology. 332 (2), 408-417 (2009).</li><li>Zou, J., Beermann, F., Wang, J., Kawakami, K., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fugu+tyrp1+promoter+directs+specific+GFP+expression+in+zebrafish:+tools+to+study+the+RPE+and+the+neural+crest-derived+melanophores.">The Fugu tyrp1 promoter directs specific GFP expression in zebrafish: tools to study the RPE and the neural crest-derived melanophores.</a> Pigment Cell Research. 19 (6), 615-627 (2006).</li><li>Xu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+into+zebrafish+embryos.">Microinjection into zebrafish embryos.</a> Methods in Molecular Biology. 127, 125-132 (1999).</li><li>Hermanson, S., Davidson, A. E., Sivasubbu, S., Balciunas, D., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleeping+Beauty+transposon+for+efficient+gene+delivery.">Sleeping Beauty transposon for efficient gene delivery.</a> Methods in Cell Biology. 77, 349-362 (2004).</li><li>Stainier, D. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+morpholino+use+in+zebrafish.">Guidelines for morpholino use in zebrafish.</a> PLoS Genetics. 13 (10), 1007000 (2017).</li><li>Wu, 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=The+progress+of+CRISPR/Cas9-mediated+gene+editing+in+generating+mouse/zebrafish+models+of+human+skeletal+diseases.">The progress of CRISPR/Cas9-mediated gene editing in generating mouse/zebrafish models of human skeletal diseases.</a> Computational and Structural Biotechnology Journal. 17, 954-962 (2019).</li><li>Affenzeller, S., Frauendorf, H., Licha, T., Jackson, D. J., Wolkenstein, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+eumelanin+and+pheomelanin+markers+in+diverse+biological+samples+by+HPLC-UV-MS+following+solid-phase+extraction.">Quantitation of eumelanin and pheomelanin markers in diverse biological samples by HPLC-UV-MS following solid-phase extraction.</a> PLoS One. 14 (10), 0223552 (2019).</li><li>Fernandes, B., Matam&#225;, T., Guimar&#227;es, D., Gomes, A., Cavaco-Paulo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+quantification+of+melanin.">Fluorescent quantification of melanin.</a> Pigment Cell &amp; Melanoma Research. 29 (6), 707-712 (2016).</li><li>Hultman, K. A., Johnson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+direct-developing+and+stem+cell-derived+melanocytes+to+the+zebrafish+larval+pigment+pattern.">Differential contribution of direct-developing and stem cell-derived melanocytes to the zebrafish larval pigment pattern.</a> Developmental Biology. 337 (2), 425-431 (2010).</li><li>Mort, R. L., Jackson, I. J., Patton, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+melanocyte+lineage+in+development+and+disease.">The melanocyte lineage in development and disease.</a> Development. 142 (4), 620-632 (2015).</li></ol>

All authors declare no conflict of interest.

Pigmentation phenotype is often manifested as alterations in the content of melanin or the number of pigment-bearing melanocytes. The method described herein allows the dissection of this dichotomy and permits qualitative as well as quantitative assessment of melanin content and the number of melanophores per embryo, irrespective of the melanin content. The high fecundity of zebrafish, visible nature of pigmented melanocytes, and lack of melanosome transfer enable the dissection of melanocyte biology using this reverse g...

While the use of melanin for photoprotection has evolved several times across the animal kingdom, vertebrates have seemingly perfected the process. Dedicated pigment-producing cells with an elaborate machinery to synthesize and contain melanin are conserved from fish to humans1. However, the outcome of pigmentation is dramatically varied, ranging in the color to recipience and presents as vivid patterns on integuments, the skin, and hair2. Despite the diversity, the repertoire of genes involved in pigmentation response is strikingly conserved. The core components of the melanin-synthesizing machinery, such as the key melanin-synthesizing enzymes, the components of the melanosomes, and the upstream connectivity to the signaling pathway, remain essentially identical across organisms. Subtle genetic differences bring about dramatic changes in the patterns of pigmentation observed across species3. Hence, a reverse genetic approach in a lower vertebrate organism, the zebrafish (Danio rerio), offers an excellent opportunity to decipher the involvement of genes in rendering the pigmented state4.
Zebrafish embryos develop from a single-celled fertilized zygote to a larva within a span of ~24 h post fertilization (hpf)5. Strikingly, the melanocyte-equivalent cells-the melanophores-are large cells that are present in the dermis and are conspicuous due to the dark melanin content6. These neural crest-derived cells emanate ~11 hpf and begin to pigment ~24 hpf6,7. Conserved gene expression modules have enabled the identification of key factors that orchestrate melanocyte functions and led to the development of transgenic fluorescent reporter lines Tg(sox10:GFP), Tg(mitfa:GFP), and Tg(ftyrp1:GFP)8,9,10 that label selective stages of melanocyte development. Using these transgenic fish lines enables the interrogation of cell biology of melanocytes at the organismal level in the tissue context with appropriate cues according to the developmental timelines. These reporters complement pigment-based quantitation of melanocytes and enable a distinct assessment of melanocyte numbers irrespective of melanin content.
This article provides a detailed protocol for deciphering the biology of melanocytes by assessing two critical parameters, namely melanin content and melanocyte numbers. While the former is a common functional readout emanating from a hypopigmentation response, the latter is associated with a reduction in the specification or survival of melanocytes and is often associated with genetic or acquired depigmentation conditions. The overall strategy of this reverse genetic screen is to silence select genes using a morpholino and investigate the melanocyte-specific outcomes. Melanin content is analyzed using image-based quantitation of mean grey values followed by confirmation using a melanin content assay. The number of melanocytes at various stages of maturation is analyzed using image-based quantitation and further confirmed using FACS analysis. Here, the screening protocol is demonstrated using two candidate genes, namely carbonic anhydrase 14, involved in melanogenesis, and a histone variant H2AFZ.2 involved in the specification of melanocytes from the neural crest precursor population. While the former alters melanin content and not the melanocyte numbers, the latter alters the number of specified melanocytes and, consequently, the melanin content in the embryo. In all, this method provides a detailed protocol to identify the role of a candidate gene in pigmentation and distinguish its role in controlling melanocyte numbers versus melanin content.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Microtubes</td>
			<td>Axygen</td>
			<td>MCT-150-A</td>
			<td>For preparing MO solution</td>
		</tr>
		<tr>
			<td>2 mL Microtubes</td>
			<td>Axygen</td>
			<td>MCT-200-C</td>
			<td>For washing steps in FACS protocol</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A-9539-500G</td>
			<td>For microinjection</td>
		</tr>
		<tr>
			<td>BD FACSAria II</td>
			<td>BD Biosciences</td>
			<td>NA</td>
			<td>For cell sorting</td>
		</tr>
		<tr>
			<td>Capillary tube</td>
			<td>Drummond</td>
			<td>1-000-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning cell strainer</td>
			<td>Corning</td>
			<td>CLS431751</td>
			<td>For making single cell suspension</td>
		</tr>
		<tr>
			<td>DMEM High Glucose Media</td>
			<td>Sigma-Aldrich</td>
			<td>D5648</td>
			<td>FACS protocol</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521-50G</td>
			<td>to immobilize ZF for imaging</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E5134</td>
			<td>For cold lysis buffer</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>BD-Biosciences</td>
			<td>342065</td>
			<td>FACS protocol</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Invitrogen</td>
			<td>10270</td>
			<td>FACS protocol</td>
		</tr>
		<tr>
			<td>Graphpad prism Software</td>
			<td>Graphstats Technologies</td>
			<td>NA</td>
			<td>For data representation</td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>National Institute of health</td>
			<td>NA</td>
			<td>For image analysis</td>
		</tr>
		<tr>
			<td>Insulin Syringes (1 mL)</td>
			<td>DispoVan</td>
			<td>NA</td>
			<td>For manual dechorionation</td>
		</tr>
		<tr>
			<td>Melanin, Synthetic</td>
			<td>Sigma-Aldrich</td>
			<td>M8631</td>
			<td>For melanin content assay</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M7027-250G</td>
			<td>to immobilize ZF for imaging</td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td>For microinjection</td>
		</tr>
		<tr>
			<td>Morpholino</td>
			<td>Gene-tools</td>
			<td>NA</td>
			<td>For knock-down experiments</td>
		</tr>
		<tr>
			<td>N-Phenylthiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td>to inhibit melanin formation</td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Sutter Instrument</td>
			<td>P-97</td>
			<td>For microinjection</td>
		</tr>
		<tr>
			<td>Nunc 15 mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>339650</td>
			<td>FACS protocol</td>
		</tr>
		<tr>
			<td>Petridish (60 mm)</td>
			<td>Tarsons</td>
			<td>460090</td>
			<td>For embryo plates</td>
		</tr>
		<tr>
			<td>Phenylmethylsulphonyl fluoride</td>
			<td>Sigma-Aldrich</td>
			<td>10837091001</td>
			<td>For cold lysis buffer</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (PBS)</td>
			<td>HiMedia</td>
			<td>TL1099-500mL</td>
			<td>For washing cells</td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Sigma-Aldrich</td>
			<td>53702</td>
			<td>For dechorionation</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8340</td>
			<td>For cold lysis buffer</td>
		</tr>
		<tr>
			<td>Sheath fluid</td>
			<td>BD FACSFlowTM</td>
			<td>342003</td>
			<td>FACS protocol</td>
		</tr>
		<tr>
			<td>Sodium phosphate</td>
			<td>Merck</td>
			<td>7558-79-4</td>
			<td>Cold lysis buffer</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284-500ML</td>
			<td>For cold lysis buffer</td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Gibco</td>
			<td>1677119</td>
			<td>For trypsinization</td>
		</tr>
	</tbody>
</table>

reverse genetic approach to identify regulators of pigmentation using zebrafish

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. 
Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.

The workflow described in Figure 1 was used to perform morpholino-based genetic perturbation at the zebrafish one-cell stage. Pigmentation analysis was performed using various methods, as mentioned below. To illustrate the representative results, standardized volumes of antisense morpholino targeting h2afv and ca14 genes were injected in the yolk or one-cell stage of the zebrafish embryo. The initial phenotyping based on brightfield imaging was performed at 48 hours post fe...

Watch this Scientific Journal Video about Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish at JoVE.com

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number.

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from ...

This protocol is an amalgamation of various techniques that would enable the researchers to delineate the role of candidate gene in the melanocyte biology. It is a holistic approach towards achieving a logical inference. By combining various methods, confounding factors can be avoided and it can help researchers gain a substantial result.To begin the analysis, immobilize the 48 HPF embryos with 0.016%tricaine. Mount the embryos using a few milliliters of 1.5 to 2%methyl cellulose in a Petri dish. Pick the embryos with a Pasteur pipette and place them in methyl cellulose to restrict movement during imaging.For optimal lateral or dorsal imaging, adjust their position with a magnification of more than 5X. Place the Petri dish under the microscope. Using a manipulator, adjust the fish so that all five melanocyte embryonic stripes are visible concurrently.Using the acquisition software, capture the images. If the fish awakens, place it into tricaine water until it stabilizes. Using the open tool in ImageJ software, open the image to be quantified.Select the freehand shape tool to outline the area for analysis. Select the set measurements option, click on the mean gray value and then area. To calculate the mean gray value for the selected area, click on M or go to analyze and select measure.Based on the stage of interest, transfer the embryos to a Petri dish containing 0.6 milligrams per milliliter Pronase. Using a Pasteur pipette, transfer the dechorionated embryos to a Petri dish containing plain embryo water after five to 10 minutes. With a glass Pasteur pipette, collect and transfer around 100 embryos to a two milliliter microcentrifuge tube.Discard the medium and add 200 microliters of ice cold deyolking Ringers solution. Place the tube on ice and mix the content around 20 times with a pipette to dissolve the yoke. Centrifuge the tubes at 100 G for one minute at four degrees Celsius twice, and discard the supernatant.Transfer the deyolked embryos using a one milliliter pipette in a Petri dish containing 10 milliliters of trypsin solution. Mix the solution once or twice using a one milliliter pipette to decrease the aggregation. Pass the trypsinized suspension through a 70 micrometer strainer placed on a 50 milliliter conical tube to collect a single cell suspension.Wash the Petri dishes with the trypsinized suspension to remove the adhered cells from the surface. For counting the fluorescently labeled cells using a flow cytometer, after creating a new experiment folder, draw a forward versus side scatter plot. Make a histogram for the intensity of fluorescein isothiocyanate.Load the wild type cells first to set the forward and side scatter gates and the FITC threshold. Afterward, load the cells isolated from transgenic FTYRP GFP line embryos to count the melanocytes. Mount the embryos in 1.5 to 2%methyl cellulose in a petri dish.Place it under the microscope and adjust it in the desired orientation using a pipette tip. Using the acquisition software, acquire the images. For embryos less than 24 HPF, using 10X magnification, capture the whole image, while for embryos more than 24 HPF, acquire multiple scan fields and subsequently assemble them.After performing the assay, the Brightfield imaging after 48 hours revealed the presence of all five pigmented melanophore stripes. At the 48 HPF stage, the number of lateral melanophores was calculated and it was found that the histone variant h2afv morphants demonstrated a reduced number of melanophores as compared to the control. The mean gray value was measured for CA14 and h2afv morphants, which revealed that the values were higher for the variants than the control morphants.By employing a sodium hydroxide-based spectrophotometric absorption method, melanin content was quantified where the CA14 morphants showed less content than the control morphants. Fluorescence imaging was performed to evaluate morpholino-based alteration for different stages of zebrafish melanophore development. The number of melanophores in CA14 and h2afv morphants was analyzed using FACS.It was observed that the number of melanophores in CA14 remained unchanged, while they were significantly reduced in h2afv compared to the control morphant. The mean fluorescence intensity per area was also calculated, demonstrating that the h2afv morphants show a considerable decrease in the value relative to control morphants. While adjusting the fish before imaging, make sure you can visualize all five melanophore stripes.You need to tilt the fish a little to distinguish between the two lateral stripes. After establishing the role of a candidate gene in melanocyte development, we can eliminate the gene in a tissue-specific way using CRISPR technology. That will be a more targeted approach.

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Biology

2.1K Görüntüleme.  CSIR-Institute of Genomics and Integrative Biology. Bu protokol, araştırmacıların melanosit biyolojisinde aday genin rolünü tanımlamalarını sağlayacak çeşitli tekniklerin bir birleşimidir. Mantıksal bir çıkarım elde etmeye yönelik bütüncül bir yaklaşımdır. Çeşitli yöntemleri birleştirerek, kafa karıştırıcı faktörlerden kaçınılabilir ve araştırmacıların önemli bir sonuç elde etmelerine yardımcı olabilir.Analize başlamak için, 48 HPF embriyosunu% 0.016 trikain ile hareketsiz hale getirin. Emb...