Name: affinity labeling detection of endogenous receptors from zebrafish embryos
Uploaded: 2016-08-31T15:00:00
Description: Watch this Scientific Journal Video about Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos at JoVE.com

The authors thank Gilberto Morales for fish care and maintenance, and Drs. Claudia Rivera and Hector Malag&#242;n (IFC-UNAM Animal Facility) for their help in rabbit immunization. This work was supported by grants from CONACYT 131226 and PAPIIT-DGAPA-UNAM IN204916.

All experiments carried out in animals were approved by the Committee for Laboratory Animal Care and Use of the Autonomous National University of M&#233;xico (UNAM), under the CICUAL-Protocol number: FLC40-14. (CICUAL: &#34;Comit&#233; Institucional para el Cuidado y Uso de los Animales de Laboratorio del Instituto de Fisiolog&#236;a Celular, Universidad Nacional Aut&#242;noma de M&#233;xico&#34;).
1. Preparation of Embryo Protein Extract
<ol>
	<li>Collect 100 - 200 embryos for each conditio...

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Chem. 263 (32), 16984-16991 (1988).</li><li>Kamaid, A., 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=Betaglycan+knock-down+causes+embryonic+angiogenesis+defects+in+zebrafish.">Betaglycan knock-down causes embryonic angiogenesis defects in zebrafish.</a> Genesis. 53, 583-603 (2015).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rapid+and+Sensitive+Method+for+the+Quantitation+of+Microgram+Quantities+of+Protein+Utilizing+the+Principle+of+Protein-Dye+Binding.">A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding.</a> Anal. 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L., Massagu&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Betaglycan+presents+ligand+to+the+TGF&#946;+signaling+receptor.">Betaglycan presents ligand to the TGF&#946; signaling receptor.</a> Cell. 73 (7), 1435-1444 (1993).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> PNAS USA. 76 (9), 4350-4354 (1979).</li><li>Maccari, F., Volpi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+specific+recognition+of+glycosaminoglycans+by+antibodies+after+their+separation+by+agarose+gel+electrophoresis+and+blotting+on+cetylpyridinium+chloride-treated+nitrocellulose+membranes.">Direct and specific recognition of glycosaminoglycans by antibodies after their separation by agarose gel electrophoresis and blotting on cetylpyridinium chloride-treated nitrocellulose membranes.</a> Electrophoresis. 24 (9), 1347-1352 (2003).</li><li>Volpi, N., Maccari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosaminoglycan+blotting+and+detection+after+electrophoresis+separation.">Glycosaminoglycan blotting and detection after electrophoresis separation.</a> Methods Mol Biol (Clifton, N.J). 1312, 119-127 (2015).</li><li>Guesdon, J. 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Biochem. 165 (2), 448-455 (1987).</li><li>Das, M., 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=Specific+radiolabeling+of+a+cell+surface+receptor+for+epidermal+growth+factor.">Specific radiolabeling of a cell surface receptor for epidermal growth factor.</a> PNAS USA. 74 (7), 2790-2794 (1977).</li><li>Massague, J., Guillette, B., Czech, M., Morgan, C., Bradshaw, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+nerve+growth+factor+receptor+protein+in+sympathetic+ganglia+membranes+by+affinity+labeling.">Identification of a nerve growth factor receptor protein in sympathetic ganglia membranes by affinity labeling.</a> J. Biol. Chem. 256 (18), 9419-9424 (1981).</li><li>Massague, J., Pilch, P. F., Czech, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+resolution+of+three+major+insulin+receptor+structures+with+unique+subunit+stoichiometries.">Electrophoretic resolution of three major insulin receptor structures with unique subunit stoichiometries.</a> PNAS USA. 77 (12), 7137-7141 (1980).</li><li>Hoosein, N. M., Gurd, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Glucagon+Receptors+in+Rat+Brain.">Identification of Glucagon Receptors in Rat Brain.</a> PNAS USA. 81, 4368-4372 (1984).</li></ol>

The use of Western blots with a specific antibody against a protein of interest is a valuable tool to study its expression7 during embryogenesis. However, immunoblotting of highly-glycosylated proteins has not been very successful due to their inefficient transfer and weak binding to nitrocellulose or PVDF membranes8,9.
Proteoglycans are a good example of this shortcoming, because of their covalently attached glycosaminoglycan chains (GAG) that are negatively charged and ...

Specific detection of proteins expressed during embryonic development is required to validate expression profiles obtained by measuring their cognate mRNAs with RT-PCR or in situ hybridization (ISH). This is commonly achieved by a western blot of embryo extracts followed by detection with specific antibodies. However, this approach is hard to apply to proteins that are in very low abundance, or that have properties that hamper their quantitative transfer during their blotting. Betaglycan, also known as the type III transforming growth factor &#946; (TGF-&#946;) receptor (TGFBR3), is an example of these difficulties. TGFBR3 is a part time membrane proteoglycan that binds TGF-&#946; through its core protein1, with notably higher affinity for the isoform TGF-&#946;2, a property that distinguishes it from any other TGF-&#946; binding protein2. TGFBR3 in the zebrafish is expressed from 8 hpf on, reaching a maximum by 72 hpf, as detected by RT-PCR of its mRNA3. 
However, despite the availability of a very specific antibody3, every attempt to detect its translated product by western blot proved unsuccessful. Reckoning that TGFBR3's proteoglycan nature, as well as putative low abundance may be accountable for this failure, a detection method, AFLIP, which takes advantage of TGFBR3 high affinity for TGF-&#946;2 was devised. In this method a protein extract from pooled embryos is allowed to specifically bind 125I-labeled TGF-&#946;2 and the receptor-ligand complexes are purified by immunoprecipitation and cross-linked before separation by SDS-PAGE. The migration patterns observed by autoradiography of the gels revealed the presence and nature of the labeled receptor species. This approach combines the ligand specificity of affinity labeling with immunoprecipitation by specific antibodies, increasing detection range, avoiding the inefficient transfer blotting of TGFBR3. Due to its inherent properties, the AFLIP assay is not a quantitative assay but can be used to confidently gauge relative experimental differences in the analyzed receptor.

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affinity labeling detection of endogenous receptors from zebrafish embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

Figure 1 shows a representative result obtained with AFLIP. Signals in Lane 1 come from the 125I-ligand covalently linked to either the zebrafish betaglycan core protein (BG core, below the 150 kDa marker) or the BG core that has been processed to its proteoglycan form by attachment of glycosaminoglycans (GAG, smear ranging from 170 kDa to the top of the gel). This pattern of migration, a sharp core protein plus a smeared proteoglycan (due to heterogeneity in t...

Watch this Scientific Journal Video about Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos at JoVE.com

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

The overall goal of this procedure is to detect low abundance growth factor receptors in zebrafish embryos by combining the selectivity and sensitivity of affinity labeling of the chosen receptor by its radioactive ligand. This method can help answer questions in developmental biology fields such as receptor expression levels and their biochemical properties. This method which we have nicknamed AFLIP extend a powerful descriptive qualities of affinity labeling, a well-established cell biology technique to zebrafish developmental studies.To begin this procedure, collect 100 to 200 embryos for each condition and place them in petri dishes containing fish water. Then manually dechorionate the embryos using fine-tipped forceps and avoid using pronase or any other protease throughout the protocol as it may digest the targeted receptors. Next, place the embryos in a 1.5-milliliter microfuge tube and wash them twice with 1X PBS.Then add 500 microliters of deyolking buffer. Release most of the yolk by gently pipetting the embryos in the solution up and down about 30 to 40 times. For 72 HPF and 48 HPF embryos, use blue and yellow pipette tips respectively.Yellow tips may need to be slightly cut to avoid destroying the embryos. A successful yolk release can be checked by observing the embryos under the microscope. Removal of yolk must be as complete as possible as yolk lipids and proteins may interfere with subsequent ligand or antibody binding steps.After that, collect the embryos by centrifugation at 600 x g for 15 seconds. Carefully discard the supernatant using a pipette. Next, wash the embryos twice with 500 microliters of washing buffer by gently vortexing them at the lowest speed.Then collect the embryos by centrifugation at 600 x g for 15 seconds. From here onwards, continue the procedure at four degrees Celsius. Discard the supernatant carefully using a pipette.Afterward, we suspend the embryos in 350 microliters of lysis buffer and homogenize them using a plastic pestle. Then incubate the lysed embryos for 30 minutes with agitation on a test tube rocker at four degrees Celsius. Next, centrifuge the lysed embryos at 11, 000 x g for 15 minutes in order to remove the insoluble debris.Following that, transfer the cleared supernatant to a fresh tube. Determine the total protein by the Bradford protein assay and plotty calibration curve. In this step, place 400 to 500 micrograms of total embryo protein in a 1.5 milliliter microfuge tube and dilute it to one microgram per microliter with buffer 1.Next, add Iodine-125 label TGF-beta II stock to reach a final concentration of 150 picomolar and incubate with agitation on a test tube rocker for two hours at four degrees Celsius. After that, add undiluted antibody against the receptor of interest in order to reach a 1:100 dilution. Then continue the incubation at four degrees Celsius for another two hours or overnight.After incubation, add 50 microliters of suitable immunoglobulin binding beads and incubate for 50 minutes with agitation on a test tube rocker at four degrees Celsius. Then recover the beads by microcentrifugation at 11, 000 x g for 20 seconds and discard the supernatant in an appropriate radioactive trash container. Subsequently, wash the IP beads three times with one milliliter of IP wash buffer by vortexing at microcentrifugation at 11, 000 x g for 20 seconds each time.Then resuspend the IP beads in 250 microliters of IP wash buffer. Prepare fresh DSS solution and add 1.5 microliters of it to the sample. Following this, incubate the sample for 15 minutes at four degrees Celsius with agitation.In order to quench the cross-linking reaction, add 500 microliters of IP wash buffer supplemented with enough tryst chloride stock at pH 7.4 to reach 25 millimolar tryst chloride. Next, centrifuge at 11, 00 x g for 20 seconds to collect IP beads. Afterward, discard the supernatant and resuspend the IP beads in 30 microliters of reducing laemmli buffer.Next, boil the sample at 94 degrees Celsius for five minutes before SDS-PAGE gel electrophoresis and then analyze the dried gels in a phosphorimeter or by conventional autoradiography. This figure shows the representative results obtained with AFLIP. Total protein extracted from the embryos at 72 hpf and 48 hpf were subjected to Iodine-125 TGF-beta II affinity labeling followed by IP with rabbit anti-zebrafish BG or rabbit anti-rat BG as a control.In the autoradiography, BG can be detected without GAG or as a glycosaminoglycan-modified BG.This figure shows how AFLIP can be used to gauge the expression levels of a given receptor in embryos subjected to an experimental manipulation, in this case, the decrease of betaglycan to the morpholino injection. Lane one shows the level of TGF beta receptor III in a 72 hpf wild-type embryo, while lane two shows the effect of administering seven nanograms of a morpholino directed to the exon 2-intron 2 boundary of the ZFPG primary transcript. Note that the BG downregulation obtained with this morpholino is not reproduced by its mismatched control.These results confirmed that the phenotype obtained with this morpholino was specific to knockdown of the TGF beta receptor III as described before. The success of AFLIP for detecting a growth factor or cytokine receptors depends on the availability of a radiolabeled ligand that can be bound and cross-linked to its receptor. A good antibody for immunoprecipitation is also require.Once mastered, this technique can be done in two days if it is performed properly. Since AFLIP avoids the transfer step of the western blot, it allows for an enhanced detection of heavily glycosylated receptors. And we have seen here for the proteoglycan betaglycan which transfer very inefficiently in western blot Following this procedure, other methods like enzymatic digestion can be performed in order to describe additional post-translational modification as long as they are revealed by electrophoretic techniques.After watching this video, you should have a good understanding of how AFLIP can help you detect low abundant receptors in several fish embryos by affinity labeling linked to immunoprecipitation. Finally, don't forget that working with Iodine-125 must be done with care and adequate protection. Good luck with your experiments.

affinity-labeling-detection-endogenous-receptors-from-zebrafish

Research

JoVE Journal

Developmental Biology

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.

A method for fluorescent staining of fixed biological material with the specific DNA label methyl green is described. Methyl green is used in a diluted aqueous solution and is very resistant to photobleaching. Its far-red emission allows for deep specimen imaging, making it particularly adequate for whole embryos.

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

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 ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

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 ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...