Name: Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish (Video) | JoVE Journal
Uploaded: 2025-01-24T14:40:00.000+00:00
Duration: 9 min 38 s
Description: Learn fluorescence labeling to visualize low-expressed proteins in zebrafish larvae. This protocol streamlines donor construction and enhances imaging of low-abundance proteins for developmental and protein research.

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5 minutes

We thank Lu Zhou (Xiamen University) for proofreading and editing this manuscript. This work was supported by the National Natural Science Foundation of China (grant 82388201 to J.H.; grant 31801158 to Y.Z.), the National Key R&#38;D Program of China (2020YFA0803500 to J.H.), the CAMS Innovation Fund for Medical Sciences (CIFMS) (2019-I2M-5-062 to J.H.), the Fujian Province Central to Local Science and Technology Development Special Program (2022L3079 to J.H.), and the FuXia-Quan Zi-Chuang District Cooperation Program (3502ZCQXT2022003 to J.H.). The funders had no role in study design, data collection and analysis, publication decisions, or manuscript preparation.

Fluorescent Protein Tagging in Zebrafish Larvae Using CRISPR/Cas9 Knock-In

<ol><li>Brittijn, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+development+and+regeneration%3A+new+tools+for+biomedical+research%5BTitle%5D)+AND+%22Int+J+Dev+Biol%22%5BJournal%5D)">Zebrafish development and regeneration: new tools for biomedical research.</a> Int J Dev Biol. 53 (5-6), 835-850 (2009).</li>
<li>Gemberling, M., Bailey, T. J., Hyde, D. R., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+zebrafish+as+a+model+for+complex+tissue+regeneration%5BTitle%5D)+AND+%22Trends+Genet%22%5BJournal%5D)">The zebrafish as a model for complex tissue regeneration.</a> Trends Genet. 29 (11), 611-620 (2013).</li>
<li>Haraoka, Y., Miyake, M., Ishitani, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+imaging+reveals+hidden+oncogenic-normal+cell+communication+during+primary+tumorigenesis%5BTitle%5D)+AND+%22Cell+Struct+Funct%22%5BJournal%5D)">Zebrafish imaging reveals hidden oncogenic-normal cell communication during primary tumorigenesis.</a> Cell Struct Funct. 48 (1), 113-121 (2023).</li>
<li>Gomes, M. C., Mostowy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+case+for+modeling+human+infection+in+zebrafish%5BTitle%5D)+AND+%22Trends+Microbiol%22%5BJournal%5D)">The case for modeling human infection in zebrafish.</a> Trends Microbiol. 28 (1), 10-18 (2020).</li>
<li>Trede, N. S., Langenau, D. M., Traver, D., Look, A. T., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+zebrafish+to+understand+immunity%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">The use of zebrafish to understand immunity.</a> Immunity. 20 (4), 367-379 (2004).</li>
<li>Karlsson, J., von Hofsten, J., Olsson, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generating+transparent+zebrafish%3A+a+refined+method+to+improve+detection+of+gene+expression+during+embryonic+development%5BTitle%5D)+AND+%22Mar+Biotechnol+%28NY%29%22%5BJournal%5D)">Generating transparent zebrafish: a refined method to improve detection of gene expression during embryonic development.</a> Mar Biotechnol (NY). 3 (6), 522-527 (2001).</li>
<li>White, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2 (2), 183-189 (2008).</li>
<li>Antinucci, P., Hindges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+crystal-clear+zebrafish+for+in+vivo+imaging%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">A crystal-clear zebrafish for in vivo imaging.</a> Sci Rep. 6, 29490(2016).</li>
<li>He, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+ligase+3-dependent+microhomology-mediated+end+joining+repair+of+DNA+double-strand+breaks+in+zebrafish+embryos%5BTitle%5D)+AND+%22Mutat+Res%22%5BJournal%5D)">Efficient ligase 3-dependent microhomology-mediated end joining repair of DNA double-strand breaks in zebrafish embryos.</a> Mutat Res. 780, 86-96 (2015).</li>
<li>Hisano, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Precise+in-frame+integration+of+exogenous+DNA+mediated+by+CRISPR%2FCas9+system+in+zebrafish%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish.</a> Sci Rep. 5, 8841(2015).</li>
<li>Luo, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR%2FCas9-based+genome+engineering+of+zebrafish+using+a+seamless+integration+strategy%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.</a> FASEB J. 32 (9), 5132-5142 (2018).</li>
<li>Wierson, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+targeted+integration+directed+by+short+homology+in+zebrafish+and+mammalian+cells%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Efficient targeted integration directed by short homology in zebrafish and mammalian cells.</a> Elife. 9, e53968(2020).</li>
<li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimal+tagging+strategies+for+illuminating+expression+profiles+of+genes+with+different+abundance+in+zebrafish%5BTitle%5D)+AND+%22Commun+Biol%22%5BJournal%5D)">Optimal tagging strategies for illuminating expression profiles of genes with different abundance in zebrafish.</a> Commun Biol. 6 (1), 1300(2023).</li>
<li>Li, C., Evans, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ligation+independent+cloning+irrespective+of+restriction+site+compatibility%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Ligation independent cloning irrespective of restriction site compatibility.</a> Nucleic Acids Res. 25 (20), 4165-4166 (1997).</li>
<li>Yu, C., Zhang, Y., Yao, S., Wei, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+PCR+based+protocol+for+detecting+indel+mutations+induced+by+TALENs+and+CRISPR%2FCas9+in+zebrafish%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A PCR based protocol for detecting indel mutations induced by TALENs and CRISPR/Cas9 in zebrafish.</a> PLoS One. 9 (6), e98282(2014).</li>
<li>Hirata, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Connexin+39.9+protein+is+necessary+for+coordinated+activation+of+slow-twitch+muscle+and+normal+behavior+in+zebrafish%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Connexin 39.9 protein is necessary for coordinated activation of slow-twitch muscle and normal behavior in zebrafish.</a> J Biol Chem. 287 (2), 1080-1089 (2012).</li>
<li>Cason, N., White, T. W., Cheng, S., Goodenough, D. A., Valdimarsson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+cloning%2C+expression+analysis%2C+and+functional+characterization+of+connexin44.1%3A+a+zebrafish+lens+gap+junction+protein%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Molecular cloning, expression analysis, and functional characterization of connexin44.1: a zebrafish lens gap junction protein.</a> Dev Dyn. 221 (2), 238-247 (2001).</li>
<li>Urushibata, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+developmental+speed+in+zebrafish+embryos+using+different+incubation+temperatures%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">Control of developmental speed in zebrafish embryos using different incubation temperatures.</a> Zebrafish. 18 (5), 316-325 (2021).</li>
<li>Yu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cyclic+digestion+and+ligation-mediated+PCR+used+for+flanking+sequence+walking%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Cyclic digestion and ligation-mediated PCR used for flanking sequence walking.</a> Sci Rep. 10 (1), 3434(2020).</li>
</ol>

All authors declare no competing interests in this paper.

To conduct this experiment smoothly and successfully, one should pay more attention to some important aspects. The optimal zebrafish developmental stage for injection should be the one-cell stage. Completing injection within this stage can ensure that all cells produced through subsequent division contain the necessary components for KI, thereby increasing the probability of simultaneous KI occurrence in all cells and improving the overall success rate of KI. Lowering the ambient temperature would slow down zebrafish emb...

As a well-accepted model organism, zebrafish (Danio rerio) is widely used in scientific research on development, regeneration, tumorigenesis, infection and immunity, etc1,2,3,4,5. Zebrafish are especially suitable for in vivo live imaging when treated with PTU (1-phenyl 2-thiourea), a toxic chemical compound that inhibits melanogenesis and makes zebrafish larvae transparent for a relatively long period of time6. Genetically modified transparent zebrafish strains, such as the casper7 and the crystal8, are also available, allowing for live visualization and lineage tracing till adulthood. Besides these efforts to overcome pigmentation for better live visualization in adult fish, our research also focuses on developing powerful gene editing strategies to make fluorescent protein tagging more efficient and less artificial.
CRISPR/Cas9-mediated microhomology-mediated end joining (MMEJ)-based KI technology is now widely applied in genetic modifications in multiple organisms, including zebrafish, due to its relatively high KI efficiency9,10,11,12. To achieve successful in vivo imaging, two challenges need to be addressed. One is the indels or scars introduced by the process of recombination; the other one is that some target genes are endogenously expressed at a low level, leading to weak signals of the co-expressed fluorescence tag and the failure of in vivo detection. Aiming for efficient and accurate protein tagging and minimizing the interference of endogenous gene expression levels, a recent work reported an optimized strategy combining a fluorescence-signal-amplifying &#34;VH&#34; tagging method and a refined germline screen workflow13, which is more suitable for fluorescence labeling low expressed genes and more efficient than previous methods by optimizing the donor backbone and combining polymerase chain reaction (PCR) and fluorescence verifications to implement germline screen at the lowest cost of time and effort. Here, we present a detailed step-by-step protocol for the reported method, including steps of embryo preparation and zebrafish husbandry, sgRNA design, in vitro transcription of sgRNAs, in vitro transcription of Cas9 mRNA, zebrafish embryo microinjection, in vivo screen for sgRNAs with the highest efficiency, donor plasmid design and construction, KI zebrafish genotyping, KI germline screen, and KI zebrafish imaging and phenotyping. This all-in-one protocol, providing a state-of-the-art fluorescence labeling method, would no doubt promote studies using zebrafish in various scientific fields and benefit readers from different academic backgrounds.

<table><tbody><tr><td>1000 &amp;micro;L&amp;nbsp; tips</td><td>Any brand</td><td>N/A</td><td>For all manipulations</td></tr><tr><td>1.5 mL tubes</td><td>Any brand</td><td>N/A</td><td>For storage and centrifugation of various solutions</td></tr><tr><td>1.5 mL tubes (RNase-free)</td><td>Any brand</td><td>N/A</td><td>For storage and centrifugation of RNase-free solutions</td></tr><tr><td>10 &amp;micro;L tips</td><td>Any brand</td><td>N/A</td><td>For all manipulations</td></tr><tr><td>10 &amp;micro;L tips (RNase-free)</td><td>Any brand</td><td>N/A</td><td>For RNase-free manipulations</td></tr><tr><td>1000 &amp;micro;L&amp;nbsp; tips (RNase-free)</td><td>Any brand</td><td>N/A</td><td>For RNase-free manipulations</td></tr><tr><td>1-phenyl 2-thiourea (PTU)</td><td>Sigma</td><td>P7629</td><td>To inhibit melanogenesis and keep the larvae transparent</td></tr><tr><td>200 &amp;micro;L tips</td><td>Any brand</td><td>N/A</td><td>For all manipulations</td></tr><tr><td>200 &amp;micro;L tips (RNase-free)</td><td>Any brand</td><td>N/A</td><td>For RNase-free manipulations</td></tr><tr><td>2x Taq Plus Master Mix II (Dye Plus)</td><td>Vazyme</td><td>P213-03</td><td>PCR for sgRNA efficiency tests</td></tr><tr><td>35-mm dishes</td><td>Corning</td><td>430165</td><td>For imaging</td></tr><tr><td>6-well plates</td><td>Any brand</td><td>N/A</td><td>For rearing zebrafish individually and temporarily</td></tr><tr><td>Acc65I</td><td>NEB</td><td>R0599S</td><td>For enzymatic digestion</td></tr><tr><td>Acetic acid</td><td>Sigma</td><td>695092</td><td>For TAE buffer preparation</td></tr><tr><td>Agar</td><td>Biofroxx</td><td>8211GR500</td><td>For LB plates</td></tr><tr><td>Ampicillin, sodium salt</td><td>Sangon</td><td>A610028</td><td>For LB plates</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C5080</td><td>For embryo buffer preparation</td></tr><tr><td>Capillary tubes</td><td>Harvard Apparatus</td><td>30-0016</td><td>For geneating needles for injection</td></tr><tr><td>Capillary tubes</td><td>Any brand</td><td>ID (inside diameter) = 0.1 mm</td><td>For quantify a 1-nL droplet</td></tr><tr><td>CRISPRscan</td><td>CRISPRscan</td><td>&amp;nbsp;http://www.crisprscan.org/</td><td>For sgRNA design</td></tr><tr><td>DH5&amp;alpha;</td><td>TIANGEN</td><td>CB101</td><td>For transformation</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td>For lysis buffer and TAE buffer preparation</td></tr><tr><td>Ensembl</td><td>Ensembl</td><td>&amp;nbsp;https://asia.ensembl.org/Danio_rerio/Info/Index</td><td>To find gene information</td></tr><tr><td>Ethanol</td><td>HUSHI</td><td>64-17-5</td><td>For DNA purification and extraction</td></tr><tr><td>Exonuclease III</td><td>Takara</td><td>2170A</td><td>LIC</td></tr><tr><td>Gas-powered micro-injector</td><td>Warner Instruments</td><td>PLI-100A</td><td>For microinjection</td></tr><tr><td>Gel electrophoresis system</td><td>WIX</td><td>WIX-EP3000</td><td>For gel electrophoresis</td></tr><tr><td>Gloves</td><td>Any brand</td><td>N/A</td><td>For all manipulations</td></tr><tr><td>Glucose</td><td>sgdbio</td><td>10010518</td><td>For LB plates</td></tr><tr><td>Glycerol</td><td>Sangon</td><td>A501745</td><td>For LB plates</td></tr><tr><td>gRNA-pMD19-T</td><td>China Zebrafish Resource Center (CZRC)</td><td>CZP3</td><td>sgRNA plasmid containing sgRNA scaffold</td></tr><tr><td>KCl</td><td>Sigma</td><td>P5405</td><td>For embryo buffer preparation</td></tr><tr><td>Lithium chloride precipitation solution</td><td>Thermofisher</td><td>AM9480</td><td>Precipitation of sgRNAs, RNase-free</td></tr><tr><td>Low melting point agarose</td><td>Sangon</td><td>A600015</td><td>For preparation of 4% agarose gels</td></tr><tr><td>Magnesium sulfate</td><td>sgdbio</td><td>20025118</td><td>For LB plates</td></tr><tr><td>Metal bath</td><td>Any brand</td><td>N/A</td><td>For constant temperature incubation</td></tr><tr><td>Methylene blue</td><td>Sigma</td><td>M9140</td><td>For embryo buffer preparation</td></tr><tr><td>Microinection mold</td><td>Homemade</td><td>N/A</td><td>For generating grooves to hold embryos during microinjection</td></tr><tr><td>Microloader, tip for filling Femtotips and other glass microcapillaries, sterile, 0.5 &amp;ndash; 20 &amp;micro;L, 100 mm, light gray, 192 pcs. (2 racks &amp;times; 96 pcs.)</td><td>Eppendorf</td><td>5242956003</td><td>To Load the microinjection solution into the injection needle, RNase-free</td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>P-97</td><td>For generating needles for injection</td></tr><tr><td>Micropipettes</td><td>Eppendorf</td><td>N/A</td><td>For all manipulations</td></tr><tr><td>Microwave</td><td>Any brand</td><td>N/A</td><td>For casting injection plate and agarose gels</td></tr><tr><td>MinElute PCR Purification Kit (50)</td><td>QIAGEN</td><td>28004</td><td>To purify PCR products and digestion products, RNase-free</td></tr><tr><td>mMESSAGE mMACHINE T3 Transcription Kit</td><td>Invitrogen</td><td>AM1348</td><td>In-vitro transcription of Cas9 mRNA, RNase-free</td></tr><tr><td>N&lt;sub&gt;2&lt;/sub&gt;</td><td>Any brand</td><td>N/A</td><td>For microinjection</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S5886</td><td>For embryo buffer and lysis buffer preparation</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td>For embryo buffer preparation</td></tr><tr><td>NaOH</td><td>Sangon</td><td>A100173</td><td>For TAE buffer preparation</td></tr><tr><td>Nuclease-free water</td><td>Thermofisher</td><td>R0581</td><td>RNA related manipulations, RNase-free</td></tr><tr><td>PciI</td><td>NEB</td><td>R0655S</td><td>For enzymatic digestion</td></tr><tr><td>PCR machine</td><td>Any brand</td><td>N/A</td><td>PCR amplification</td></tr><tr><td>PCR strip tubes</td><td>Any brand</td><td>N/A</td><td>For PCR</td></tr><tr><td>PCR tubes</td><td>FEITONGKANG</td><td>blp-200</td><td>For PCR</td></tr><tr><td>Petri dish</td><td>Any brand</td><td>N/A</td><td>For rearing zebrafish larvae etc.</td></tr><tr><td>Phenol red</td><td>Sigma</td><td>P0290</td><td>For microinjection, RNase-free</td></tr><tr><td>Pipette holder</td><td>Warner Instruments</td><td>PLI-PH1</td><td>For microinjection</td></tr><tr><td>PrimeStar (Premix)</td><td>Takara</td><td>DR040A</td><td>For molecular cloning</td></tr><tr><td>Proteinase K</td><td>Merck</td><td>539480</td><td>For genomic DNA extraction</td></tr><tr><td>pT3TS (T3:zCas9-UTRglobin)</td><td>China Zebrafish Resource Center (CZRC)</td><td>CZP11</td><td>Cas9 plasmid template</td></tr><tr><td>Refrigerated centrifuge</td><td>Any brand</td><td>N/A</td><td>For RNA related centrifugation</td></tr><tr><td>Scale</td><td>Sartorius</td><td>BCE124-1CCN</td><td>For casting injection plate and agarose gels</td></tr><tr><td>Scissors</td><td>Any brand</td><td>N/A</td><td>For clipping adult fish tails</td></tr><tr><td>SDS</td><td>Sangon</td><td>A600485</td><td>For lysis buffer preparation</td></tr><tr><td>Small brushes</td><td>Any brand</td><td>N/A</td><td>For gently moving embryos on microinjection plates</td></tr><tr><td>Spectrophotometer</td><td>Thermofisher</td><td>NanoDrop 2000</td><td>For DNA/RNA concentration measurements</td></tr><tr><td>Stage micrometer</td><td>Any brand</td><td>DIV (Division) = 0.01 mm</td><td>For quantify a 1-nL droplet</td></tr><tr><td>Stereo fluorescence microscope</td><td>Olympus</td><td>SZX16</td><td>For observation of gene expression</td></tr><tr><td>Stereo microscope</td><td>Motic</td><td>SMZ-168</td><td>For observation and microinjection</td></tr><tr><td>T7 RiboMAX Express Large Scale RNA Production System</td><td>Promega</td><td>P1320</td><td>In-vitro transcription of sgRNAs, RNase-free</td></tr><tr><td>Temperature-controlled incubator</td><td>Any brand</td><td>N/A</td><td>For embryo incubation</td></tr><tr><td>TIDE</td><td>TIDE</td><td>https://tide.nki.nl/</td><td>For sgRNA efficiency analysis&amp;nbsp;</td></tr><tr><td>Tricaine methanesulfonate</td><td>Sigma</td><td>A5040</td><td>For tricaine solution preparation</td></tr><tr><td>Tris</td><td>Sangon</td><td>A600194</td><td>For tricaine solution, lysis buffer and TAE buffer preparation</td></tr><tr><td>Tryptone</td><td>OXOID</td><td>LP0042</td><td>For LB plates</td></tr><tr><td>Tweezers</td><td>Any brand</td><td>N/A</td><td>For cutting injection needles</td></tr><tr><td>XbaI</td><td>NEB</td><td>R0145S</td><td>For enzymatic digestion</td></tr><tr><td>Yeast extract</td><td>OXOID</td><td>LP0021</td><td>For LB plates</td></tr><tr><td>Zebrafish aquarium system</td><td>ESEN</td><td>ESEN-AW-S1</td><td>For rearing adult fish</td></tr><tr><td>Zebrafish mating tanks (with a divider)</td><td>Any brand</td><td>N/A</td><td>For mating of the fish</td></tr><tr><td>Zeiss LSM 900+Airyscan2</td><td>ZEISS</td><td>Zeiss LSM900</td><td>For confocal imaging of the fluorescence-labeled fish and data analysis</td></tr><tr><td>ZFIN website</td><td>ZFIN</td><td>http://zfin.org/</td><td>To find gene information</td></tr></tbody></table>

fluorescence labeling to visualize low-expressed proteins in zebrafish

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

Gap junctions consisting of polymeric proteins called connexins are essential for the exchange of low-molecular-weight metabolites and ions between contacting cells. The fluorescent protein-tagging method described above was used to label connexin 39.9 (Figure 5A) and connexin 44.1 (Figure 5B), respectively. Correct KI was verified by junction PCRs (Figure 6). In agreement with published data16</s...

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

fluorescence-labeling-to-visualize-low-expressed-proteins-in-zebrafish

Research

JoVE Journal

Genetics

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes.  We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Labeling and Imaging Cells in the Zebrafish Hindbrain

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia.  Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used.  This technique can be applied to any cell type.

Single Cell Fate Mapping in Zebrafish

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

Imaging Subcellular Structures in the Living Zebrafish Embryo

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from early-stage zebrafish embryos enabling precise colocalization analyses in specific cell populations.

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Learn fluorescence labeling to visualize low-expressed proteins in zebrafish larvae. This protocol streamlines donor construction and enhances imaging of low-abundance proteins for developmental and protein research.

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

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