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><tbody><tr><td>Disuccinimidyl suberate (DSS)</td><td>ThermoFisher Scientific</td><td>21555</td><td></td></tr><tr><td>Protein G Sepharose 4 Fast Flow</td><td>GE Healthcare Life Sciences</td><td>17-0618-01</td><td></td></tr><tr><td>Gel Dryer Model 583&amp;nbsp;</td><td>BIO-RAD</td><td>1651745</td><td></td></tr><tr><td>Typhoon 9400</td><td>GE Healthcare Life Sciences</td><td>63-0055-78</td><td></td></tr><tr><td>Cobra II Auto gamma counter</td><td>Packard</td><td></td><td></td></tr><tr><td>Exposure Cassette</td><td>Molecular Dynamics</td><td>63-0035-44</td><td></td></tr><tr><td>NaCl</td><td>J.T. Baker</td><td>3624</td><td></td></tr><tr><td>KCl</td><td>J.T. Baker</td><td>3040</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T. Baker</td><td>3828</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T. Baker</td><td>3246</td><td></td></tr><tr><td>CH&lt;sub&gt;3&lt;/sub&gt;OH</td><td>J.T. Baker</td><td>9070</td><td></td></tr><tr><td>CH&lt;sub&gt;3&lt;/sub&gt;COOH</td><td>J.T. Baker</td><td>9508</td><td></td></tr><tr><td>HCHO</td><td>J.T. Baker</td><td>2106</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L4509</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>ED</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C3306</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S233</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>P7626</td><td></td></tr><tr><td>Crystal Sea Marine Mix</td><td>Marine Enterprises International</td><td>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

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

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.

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Neuroscience

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

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

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.

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 ...

Genetics

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. ...

Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing organs of both invertebrates and vertebrates, asymmetrically dividing progenitors generates a Notchhi self-renewing and a Notchlo differentiating daughter. In the embryonic zebrafish brain, radial glia progenitors (RGPs)-the principal vertebrate neural stem cells-mostly undergo ACD to give birth to one RGP and one differentiating neuron. The optical clarity and easy accessibility of zebrafish embryos make them ideal for in vivo time-lapse imaging to directly visualize how and when the asymmetry of Notch signaling is established during ACD. Recent studies have shown that dynamic endocytosis of the Notch ligand DeltaD plays a crucial role in cell fate determination during ACD, and the process is regulated by the evolutionarily conserved polarity regulator Par-3 (also known as Pard3) and the dynein motor complex. To visualize the in vivo trafficking patterns of Notch signaling endosomes in mitotic RGPs, we have developed this antibody uptake assay. Using the assay, we have uncovered the dynamicity of DeltaD-containing endosomes during RGP division.

This work develops an antibody uptake assay for imaging intra-lineage Notch/DeltaD signaling in dividing radial glia progenitors of the embryonic zebrafish brain.

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

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.

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Biology

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

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

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

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

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

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

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

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.

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

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

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

Summary

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Chapters in this video

Labeling and Imaging Cells in the Zebrafish Hindbrain

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

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

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

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos