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 condition to compare (morphants vs. wild type) at desired stage, for example 72 hr post fertilization (hpf).</li>
	<li>Place embryos in Petri dishes containing fish water (see Table 1) and manually dechorionate embryos using fine-tipped forceps. Avoid using pronase during this step (or any other protease throughout the protocol) as it may digest the targeted receptor.</li>
	<li>Place embryos in a 1.5 ml microfuge tube and wash embryos twice with 1x phosphate buffer solution (PBS) (see Table 1).</li>
	<li>Add 500 &#181;l of deyolk buffer (see Table 1).</li>
	<li>Release most of yolk by gently pipetting up and down (about 30 - 40 times) the embryos in the solution. 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 embryos. The successful yolk release can be checked by observing embryos under microscope.</li>
	<li>Collect embryos by centrifugation at 600 x g for 15 sec.</li>
	<li>Discard supernatant carefully by using a pipet.</li>
	<li>Wash embryos twice with 500 &#181;l washing buffer (see Table 1) by gently vortexing at lowest speed.</li>
	<li>Collect embryos by centrifugation at 600 x g for 15 sec.</li>
	<li>Starting from here, continue the procedure at 4 &#176;C.</li>
	<li>Discard supernatant carefully by using a pipet.</li>
	<li>Resuspend embryos in 350 &#181;l of lysis buffer (see Table 1) and homogenize using a plastic pestle.</li>
	<li>Incubate lysed embryos 30 min with agitation on a test tube rocker at 4 &#176;C.</li>
	<li>Centrifuge lysed embryos at 11,000 x g for 15 min in order to remove insoluble debris.</li>
	<li>Transfer cleared supernatant using a pipet to a fresh tube.</li>
	<li>Determine total protein by Bradford protein assay4, or other suitable procedure. If Bradford assay is used, perform the calibration curve in the presence of equivalent concentrations of the detergents presents in the lysis buffer, as they cause an underestimation of the protein standard4.</li>
</ol>
2. Detection of Endogenous Receptor Protein
<ol>
	<li>Affinity Labeling and Immunoprecipitation (all these steps at 4 oC)
	<ol>
		<li>Place 400 - 500 &#181;g of total embryo protein in a 1.5 ml microfuge tube and dilute to 1 &#181;g/&#181;l with buffer 1 (see Table 1). 
		Note: In order to obtain 500 &#181;g of total embryo protein, about 100 - 200 embryos must be initially processed. 72 hpf embryos routinely yield ~ 5 &#181;g of total embryo protein, which is sufficient for betaglycan detection.</li>
		<li>Add enough volume of labeled TGF-&#946;2 stock to reach a final concentration of 150 pM and incubate with agitation on a test tube rocker 2 hr at 4 &#176;C. TGF-&#946;2 must be labeled before AFLIP is started, with 125I by the chloramine T method as described by Cheifetz et al.5. 
		CAUTION: Use shielding to minimize exposure while handling 125I labeled ligand.</li>
		<li>Add enough volume of undiluted antibody against the receptor of interest in order to reach a 1:100 dilution and continue incubation for another 2 hr at 4 &#176;C (incubation may be O/N). This is the optimal dilution of antiserum # 31, used in this study and described elsewhere3, but depending on the quality of the antibody, a larger or smaller dilution may be the optimal.</li>
		<li>Add 50 &#181;l of suitable immunoglobulin binding beads (e.g., G-protein-Sepharose, which was previously equilibrated in TNTE and resuspended 1:5 of its original volume in TNTE) and incubate for 50 min with agitation on a test tube rocker at 4 &#176;C.</li>
		<li>Recover the beads by microcentrifugation at 11,000 x g for 20 sec.</li>
		<li>Discard supernatant in an appropriate radioactive trash container.</li>
		<li>Wash the IP-beads three times with 1 ml of IP wash buffer (see Table 1), by vortexing and microcentrifugation at 11,000 x g for 20 sec each time.</li>
		<li>Resuspend IP-beads in 250 &#181;l of IP wash buffer.</li>
		<li>Add 1.5 &#181;l of disuccinimidyl suberate (DSS, dissolved in DMSO at 10 mg/ml). Prepare the DSS solution just before its use in this step.</li>
		<li>Incubate for 15 min at 4 &#176;C with agitation.</li>
		<li>In order to quench the crosslinking reaction, add 500 &#181;l of IP wash buffer, supplemented with enough Tris-Cl pH 7.4 stock to reach 25 mM Tris-Cl. The free amino groups in Tris capture unreacted DSS.</li>
		<li>Centrifuge at 11,000 x g for 20 sec to collect IP-beads and discard supernatant.</li>
		<li>Resuspend IP-beads in 30 &#181;l of reducing Laemmli buffer.</li>
		<li>Boil samples 5 min at 94 &#176;C.</li>
		<li>Optionally, analyze samples in a gamma counter using manufacturer&#39;s protocol.</li>
	</ol>
	</li>
	<li>Sample Analysis
	<ol>
		<li>Subject samples to denaturing SDS-PAGE. Use polyacrylamide at the appropriate percentage for the mass of the receptor and run gel under standard procedure.</li>
		<li>Immerse gel in fixative solution (see Table 1) for 30 min at RT.</li>
		<li>Wash gel with distillated water for 15 min.</li>
		<li>Place gel in previously hydrated filter paper and cover with saran wrap film.</li>
		<li>Dry gel at 80 &#176;C for 1 hr.</li>
		<li>Expose gel on a white phosphorimager screen at RT O/N.</li>
		<li>Scan exposed screen using a phosphorimager using manufacturer&#39;s protocol.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>L&#243;pez-Casillas, F., 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=Structure+and+expression+of+the+membrane+proteoglycan+betaglycan,+a+component+of+the+TGF-&#946;+receptor+system.">Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-&#946; receptor system.</a> Cell. 67 (4), 785-795 (1991).</li><li>Cheifetz, S., Andres, J. L., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transforming+growth+factor-beta+receptor+type+III+is+a+membrane+proteoglycan.+Domain+structure+of+the+receptor.">The transforming growth factor-beta receptor type III is a membrane proteoglycan. Domain structure of the receptor.</a> J. Biol. 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. Biochem. 72, 248-254 (1976).</li><li>Cheifetz, 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=Distinct+transforming+growth+factor-b+receptor+subsets+as+determinants+of+cellular+responsiveness+to+three+TGF-b+isoforms.">Distinct transforming growth factor-b receptor subsets as determinants of cellular responsiveness to three TGF-b isoforms.</a> J. Biol. Chem. 265, 20533-20538 (1990).</li><li>L&#243;pez-Casillas, F., Wrana, J. 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. L., Ternynck, T., Avrameas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+avidin-biotin+interaction+in+immunoenzymatic+techniques.">The use of avidin-biotin interaction in immunoenzymatic techniques.</a> J Histochem Cytochem. 27 (8), 1131-1139 (1979).</li><li>Bratthauer, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+avidin-biotin+complex+(ABC)+method+and+other+avidin-biotin+binding+methods.">The avidin-biotin complex (ABC) method and other avidin-biotin binding methods.</a> Methods Mol Biol (Clifton, N.J). 588, 257-270 (2010).</li><li>Heimer, R., Molinaro, L., Sampson, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+by+125I-cationized+cytochrome+c+of+proteoglycans+and+glycosaminoglycans+immobilized+on+unmodified+and+on+positively+charged+Nylon+66.">Detection by 125I-cationized cytochrome c of proteoglycans and glycosaminoglycans immobilized on unmodified and on positively charged Nylon 66.</a> Anal. 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 authors have nothing to disclose.

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 do not bind well to either polystyrene surfaces or hydrophobic blotting membranes. Also, the size heterogeneity of the GAG chains &#34;spreads&#34; the protein over a sector of the gel, effectively decreasing its amount per area of the blot. In addition, if the protein of interest has low levels of expression, its detection by regular western blots is a difficult task. The type III transforming growth factor &#946; receptor (TGFBR3) or betaglycan (BG), a membrane receptor with two GAG&#39;s attachment sites in mammals1 and one in zebrafish3, has all of these properties. Although, protocols to overcome these hurdles have been devised, like the detection by avidin-biotin complex (ABC) system10,11 or the immunobloting of proteoglycans on positively charged membranes12, they cannot resolve both problems in the same protocol. Taking advantage of the high affinity of BG for its natural ligand TGF&#946;2 and of the availability of a specific antibody, AFLIP, a technique that combines the advantages of affinity labeling and immunoprecipitation, the above discussed limitations of western blot detection may be overcome.
Affinity labeling of membrane-bound receptors with their radiolabeled ligands is a well used tool that has been instrumental for the identification and characterization of several important growth factors receptors in cultured cells13-16. While in conventional affinity labeling a monolayer of cells is subjected to covalent ligand labeling on the tissue culture dish and then lysed, in AFLIP the ligand-receptor complexes are first formed in embryo lysates, then purified by immunoprecipitation and finally, covalently cross-linked. Because of its use of radiolabeled ligands, AFLIP is very sensitive and in principle can be adapted to other growth factors or cytokines receptors for which a labeled ligand and a good antibody are available. This potential of AFLIP would help the detection of these low abundant but physiological relevant molecules.
Because of the inherent variability in the cross-linking step of affinity labeling, AFLIP cannot be used to quantitatively determine the levels of the measured receptors. However, if performed with adequate parallel controls, it can provide a quite accurate gauging of relative levels of their expression. As AFLIP use total protein extracts, it is not restricted to one specific cellular location of the receptor, it reveals its expression in the whole individual or organ, if applied to dissected adult specimens. Finally, if needed AFLIP could be linked to other biochemical protocols, like carbohydrate enzymatic digestions, to further characterize the receptor of interest.
Similar to conventional affinity labeling, a freshly 125I-labeled ligand is strongly recommended. Given the short half-life of 125I, the ligand must be used within the first 2 weeks after labeling with fresh isotope. Although most of the caveats of the protocol have been mentioned in the corresponding steps, one special mention shall be given to the careful removal of the yolk, which must be as complete and uniform as possible. Leaving uneven amounts of yolk would result in incorrect quantitation of bona fide embryo protein in the lysates.

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.

<table border="0" cellpadding="0" cellspacing="0" width="670">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Disuccinimidyl suberate (DSS)</td>
			<td style="width:168px;">ThermoFisher Scientific</td>
			<td style="width:119px;">21555</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Protein G Sephraose 4 Fast Flow</td>
			<td style="width:168px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-0618-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Gel Dryer Model 583&#160;</td>
			<td style="width:168px;">BIO-RAD</td>
			<td style="width:119px;">1651745</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Typhoon 9400</td>
			<td style="width:168px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">63-0055-78</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cobra II Auto gamma counter</td>
			<td style="width:168px;">Packard</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Exposure Cassette</td>
			<td style="width:168px;">Molecular Dynamics</td>
			<td style="width:119px;">63-0035-44</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Na2HPO4</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">K2HPO4</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3246</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CH4O</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">9070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C2H4O2</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">9508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CH2O</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">2106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">L4509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">ED</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">C3306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaHCO3</td>
			<td style="width:168px;">Fisher Scientific</td>
			<td style="width:119px;">S233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PMSF</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7626</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Crystal Sea Marine Mix</td>
			<td style="width:168px;">Marine Enterprises International</td>
			<td style="width:119px;">http://www.meisalt.com/Crystal-Sea-Marinemix</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 the length of the GAG chains), is characteristic of the TGFBR32. Since the DSS does not covalently link every single ligand-receptor complex formed, there is free 125I-ligand appearing at the migration front of the gel (125I-TGF-&#946;2). This free ligand was bound to the receptor, and remained bound during the immunoprecipitation, but since it was not covalently attached, detached during the SDS-PAGE procedure. Nonetheless, the strength of its signal still correlates to the one given by the labeled receptor. This can be appreciated by comparing lanes 2 and 3. At 48 hpf the zebrafish embryos exhibit lower amounts of BG than 72 hpf embryos3, which can be seen by the faint signals at the GAG and BG core, that correspond to the intensity of the signals given by the free ligand appearing at the front of the gel (Lane 2). Given that antibodies against rat BG6 do not cross-react with the ZF BG, the antibodies when used in the AFLIP gave no signal, therefore, serving as a negative control (Lane 3). Similar negative results were obtained with the pre-immune serum of the anti-ZF BG antibody3.
Figure 2 shows how AFLIP can be used to gauge the levels of expression of a given receptor in embryos subjected to an experimental manipulation, in this case, the decrease of betaglycan due to morpholino injection3. Lane 1 shows the level of TGFBR3 in a 72 hpf wild type embryo, while Lane 2 shows the effect of administering 7 ng of a morpholino directed to the exon2-intron2 boundary of the ZF BG primary transcript. Note that the BG down regulation obtained with this morpholino is not reproduced by its mis-matched control (Lane 3). These results confirmed that the phenotype obtained with this morpholino was specific for the knockdown of the TGFBR3 as described before3.
<img alt="Figure 1" src="/files/ftp_upload/54405/54405fig1.jpg" /> 
Figure 1. AFLIP Detection of BG in Zebrafish Embryos.Total protein extract from embryos at 72 hr post-fertilization (hpf) (Lanes 1 and 3) and 48 hpf (Lane 2) were subjected to 125I-TGF-&#946;2 affinity labeling followed by IP with rabbit anti-zebrafish BG (Lanes 1 and 2, zBG) or rabbit anti-rat BG (Lane 3, rBG) as a control. In the autoradiography BG can be detected without GAG (BG core) or as a glycosaminoglycan-modified BG (GAG). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54405/54405fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54405/54405fig2.jpg" /> 
Figure 2. BG Morpholino Knockdown Detected by AFLIP. 125I-TGF-&#946;2 affinity labeling of BG from untreated WT embryos (lane1), microinjected with specific BG morpholino (Lane 2) or a mismatch control (Lane 3). The counts per million (cpm) recovered after the immunoprecipitation and&#160;the cross-linking step are indicated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54405/54405fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer&#160;</td>
			<td>Composition</td>
		</tr>
		<tr>
			<td>Buffer 1</td>
			<td>50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100</td>
		</tr>
		<tr>
			<td>Deyolk buffer</td>
			<td>55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO3&#160;</td>
		</tr>
		<tr>
			<td>Fish water</td>
			<td>See Materials Table</td>
		</tr>
		<tr>
			<td>Fixative solution</td>
			<td>50% CH3OH, 12% CH3COOH, 0.185% HCHO</td>
		</tr>
		<tr>
			<td>IP wash buffer</td>
			<td>10 mM Na2HPO4 , 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.1% Triton X-100, 0.02% SDS, pH 7.4</td>
		</tr>
		<tr>
			<td>Laemmli buffer</td>
			<td>2% SDS, 10% Glycerol, 100 mM DTT, 60 mM Tris (pH 6.8), 0.001% bromphenol blue.</td>
		</tr>
		<tr>
			<td>Lysis buffer</td>
			<td>50 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.5% Triton X-100, 0.1% SDS, 0.5% Sodium Deoxycholate</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH7.4</td>
		</tr>
		<tr>
			<td>TNTE</td>
			<td>50 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100</td>
		</tr>
		<tr>
			<td>Washing buffer</td>
			<td>110 mM NaCl, 3.5 mM KCl, 2.7 mM CaCl2, 10 mM Tris-HCl&#160;pH 8.5</td>
		</tr>
	</tbody>
</table>
Table 1: Buffer Compositions

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

5.6K Views.  Universidad Nacional Autónoma de México. 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.

Video: Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

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. ...