We thank Dr. Jingrong Peng for sharing the Tg (fabp10a: DsRed) transgenic line; Dr. Zilong Wen for sharing the Tg (mpeg1: GFP) transgenic lines; Dr. Koichi Kawakami for providing the pTol2 vector. This work was supported by the National Natural Science Foundation of China (31771594), Guangdong Science and Technology Plan projects (2019A030317001) and the Fundamental Research Funds for the Central Universities (D2191450).

NOTE: All the samples were treated by phenylthiourea(PTU) egg water to inhibit pigment.
1. Generation of Tg (fabp10a:il34) Transgenic Constructs and Fish Injection
<ol>
	<li>Clone the 2.8 kb fabp10a promoter8 and the IL-34 coding regions (ENSDART00000126460.3) of zebrafish into the pTol2 vector to generate the fabp10a-il34 construct. Inject the constructs into one-cell stage Tg (mpeg1: GFP) and WT fish embryos together with the transposase mRNA. Raise the fabp10a-il34 injected WT embryos to adult9 and identify the transgenic founder by in situ hybridization. 
	NOTE: The injection of the Tol2 construct directly into another transgenic could be problematic if the other transgenic line is made with the same transposon system. A general practice would be to make an independent transgenic line and subsequently cross the new line with another reporter line. This ensures that there will be no effects of the new transgenesis on a previously inserted transgene.</li>
</ol>
2. Fluorescent Whole Mount In Situ Hybridization (WISH) Combine with Immunostaining
<ol>
	<li>Sample fixation
	<ol>
		<li>Collect embryos of transient injection or stable IL-34 transgenic line which crossed with Tg (mpeg1: GFP) at desired stages. 
		NOTE: For this case, the embryos were collected at 4 d post fertilization (dpf). (If needed) remove the chorion by syringe.</li>
		<li>Fix the embryos with 4% paraformaldehyde (PFA) overnight at 4 &#176;C or 2 h at room temperature (RT) (about 25 &#176;C).</li>
		<li>Wash the embryos with phosphate buffered saline plus Tween 20 (PBST) 3x 5 min.</li>
		<li>Dehydrate the embryos separately with 50% methanol in PBST (50% Methanol/PBST) and 100% methanol, 1x 5 min each. Then, change to fresh 100% methanol and store at -20 &#176;C (at least 2 h). 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
	<li>Probe hybridization (Day I)
	<ol>
		<li>Rehydrate the embryos in the previous steps with 50% methanol in PBST (50% Methanol/PBST), then wash with PBST 3x 5 min.</li>
		<li>Digest the embryos with proteinase K in PBST at RT (final concentration: 10 &#181;g/mL; 1:2000 in PBST). 
		NOTE: Digestion time depends on the embryos stage: Less than 36 h post fertilization (hpf), no need; 36 hpf-2 dpf embryo, 3-5 min; 2-3 dpf embryo, 10 min; 3-4 dpf embryo, 15 min; 4-5 dpf embryo, 15-20 min; 5-6 dpf embryo, 20-27 min; &#62;6 dpf embryo, 25-30 min at RT (about 25 &#176;C).</li>
		<li>Discard the digestion solution and perform fixation again with 4% PFA, for 20 min at RT.</li>
		<li>Wash the embryos with PBST 2x 10 min.</li>
		<li>Discard the PBST, perform pre-hybridization with heated hybridization buffer (HB buffer) at 65 &#176;C for 5 min, recycle the HB buffer into the original tube.</li>
		<li>Perform pre-hybridization with new heated HB buffer at 65 &#176;C at least 1 h.</li>
		<li>Pre-heat the probe9 (for this case was an il34 probe, 1 ng/mL) at 65 &#176;C at least 10 min. Then recycle the HB buffer into the original tube. Perform hybridization with the pre-heated probe at 65 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Antibody treatment (Day II)
	<ol>
		<li>Pre-heat the 50% formamide/2x saline sodium citrate plus Tween 20 (SSCT), 2x SSCT, 0.2x SSCT at 65 &#176;C.</li>
		<li>Recycle the probe into the original tube and store the probe at -20 &#176;C.</li>
		<li>Wash the embryos separately with 50% formamide/2x SSCT; 2x SSCT; 0.2x SSCT, 3x 20 min or 2x 30 min each at 65 &#176;C.</li>
		<li>Wash the embryos with PBST 3x 5 min.</li>
		<li>Block the samples with 600 &#181;L of blocking buffer (5% filtered fetal bovine serum (FBS) in PBST) for 1 h at RT.</li>
		<li>Add 400 &#181;L of Anti-digoxigenin-HRP antibody solution (1:1,000-1:2,000 in blocking buffer) and incubate the embryos at 4 &#176;C overnight. If signals are weak, use 1:500 dilution of antibody.</li>
	</ol>
	</li>
	<li>Coloring and primary antibody incubating (Day III)
	<ol>
		<li>Remove the antibody; wash the embryos with PBST, 6x 20 min at RT.</li>
		<li>Rinse the sample with 30 &#181;L of 1x Plus Amplification Diluent for 5 min at RT.</li>
		<li>Discard the diluent by pipetting out; dilute Fluorophore Tyramide Stock Solution (Cyanine 3 Plus Amplification Reagent (Cy3) or Cyanine 5 Plus Amplification Reagent (Cy5), for this case Cy3 was used) 1:50 in 1x Plus Amplification Diluent to make the Fluorophore Tyramide Working Solution. Prepare 50-100 &#181;L of working solution for each sample.</li>
		<li>Incubate the sample in the Fluorophore Tyramide Working Solution for 5-15 min in dark at RT. If signals are weak, extend the incubation time to 30 min.</li>
		<li>Stop the reaction by changing the working solution with PBST and examine the signals.</li>
		<li>Wash the embryos with PBST 3x 10 min at RT.</li>
		<li>Incubate the sample with primary antibody at 4 &#176;C overnight. For this case, use the Goat-Anti-GFP antibody as the primary antibody.</li>
	</ol>
	</li>
	<li>Secondary antibody staining (Day IV)
	<ol>
		<li>Wash the embryos with PBST for 4x 30 min.</li>
		<li>Incubate the embryos with secondary antibody at 4 &#176;C overnight. For this case, use Alexa 488-Anti-Goat antibody as the secondary antibody.</li>
	</ol>
	</li>
	<li>Take pictures (Day V)
	<ol>
		<li>Wash the embryos with PBST 3x 10 min at RT.</li>
		<li>Store the embryos in 70% glycerol in dark at 4 &#176;C overnight or -20 &#176;C for longer.</li>
	</ol>
	</li>
</ol>
3. Live Imaging
<ol>
	<li>Sample selecting 
	NOTE: Use the live image to directly observe whether macrophages of Tg (fabp10a: il34; fabp10a: DsRed; mpeg1: GFP) fish would migrate into the liver under IL-34 induction during 3-3.5 dpf. Here the Tg (fabp10a-DsRed) transgenic line is used to label the liver region and make it visible, to facilitate the localization of the liver and to determine whether macrophages migrate into the liver. Before imaging, use a fluorescence microscope to select the DsRed and GFP double positive embryos.</li>
	<li>Fish mounting
	<ol>
		<li>Use a metal bath to heat 1 mL of 1% low melting agarose to above 90 &#176;C to completely melt it.</li>
		<li>After the low melting agarose is cooled to body temperature, add 50 &#181;L of 0.2% tricaine, and uniformly mix tricaine with the agarose.</li>
		<li>Move the anesthetized embryos to a small dish mounted with a cover slide on the bottom, remove the surrounding water, slowly drop the low melting agarose on the embryos, carefully set the position of the fish before the agarose is solidified, keep the liver area close to the cover slide on the bottom of the dish.</li>
		<li>After the low melting agarose is solidified, carefully cover it with another layer of agarose to reinforce it.</li>
		<li>Place the dish on the confocal microscope carrier table, cover the fish with the E2 solution10 with tricaine and start imaging.</li>
	</ol>
	</li>
	<li>Software operation of the confocal microscope
	<ol>
		<li>Open the ZEN black 2.3 software, install the living cell workbench onto the microscope carrier table.</li>
		<li>Click Locate | Incubation | Temperature to set the temperature to 29 &#176;C.</li>
		<li>Place the dish in the center of the living cell workbench, cover the fish with the E2 solution10 with tricaine.</li>
		<li>Click the Acquisition menu, select the required scan mode and lasers in the Smart Setup menu, then select Z-Stack and Position.</li>
		<li>Click the Experiment Designer menu, select Enable Multi Block Experiment, in the first block, to find the sample under the low magnification, then switch to the high magnification, let the observed area in the center of the visual field.</li>
		<li>Set the position and Z-Stack information, select the appropriate laser intensity, scanning layers and imaging speed.</li>
		<li>Create a new block and repeat the above steps. After setting up all blocks, set the appropriate number of loops and start recording (Figure 1).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Howe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-34+is+a+tissue-restricted+ligand+of+CSF1R+required+for+the+development+of+Langerhans+cells+and+microglia.">IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia.</a> Nature Immunology. 13 (8), 753-760 (2012).</li><li>Lin, H., 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=Discovery+of+a+cytokine+and+its+receptor+by+functional+screening+of+the+extracellular+proteome.">Discovery of a cytokine and its receptor by functional screening of the extracellular proteome.</a> Science. 320 (5877), 807-811 (2008).</li><li>Wei, 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=Functional+overlap+but+differential+expression+of+CSF-1+and+IL-34+in+their+CSF-1+receptor-mediated+regulation+of+myeloid+cells.">Functional overlap but differential expression of CSF-1 and IL-34 in their CSF-1 receptor-mediated regulation of myeloid cells.</a> Journal of leukocyte biology. 88 (3), 495-505 (2010).</li><li>Etienne, D., Foucher, S. B. L. P., Norbert Ifrah, P. G. Y. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-34+Induces+the+Differentiation+of+Human+Monocytes+into+Immunosuppressive+Macrophages.+Antagonistic+Effects+of+GM-CSF+and+IFNc.">IL-34 Induces the Differentiation of Human Monocytes into Immunosuppressive Macrophages. Antagonistic Effects of GM-CSF and IFNc.</a> PLoS One. 8 (2), e56045 (2013).</li><li>Segaliny, A. I., 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=Syndecan-1+regulates+the+biological+activities+of+interleukin-34.">Syndecan-1 regulates the biological activities of interleukin-34.</a> Biochimica et Biophysica Acta. 1853 (5), 1010-1021 (2015).</li><li>Zhou, S. L., 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=miR-28-5p-IL-34-macrophage+feedback+loop+modulates+hepatocellular+carcinoma+metastasis.">miR-28-5p-IL-34-macrophage feedback loop modulates hepatocellular carcinoma metastasis.</a> Hepatology. 63 (5), 1560-1575 (2016).</li><li>Gordon, J. I., 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=Tissue+specific+expression+and+developmental+regulation+of+two+genes+coding+for+rat+fatty+acid+binding+proteins.">Tissue specific expression and developmental regulation of two genes coding for rat fatty acid binding proteins.</a> Journal of Biological Chemistry. 260 (4), 1995-1998 (1985).</li><li>Westerfield, 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> The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio). , (2000).</li><li>N&#252;sslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish: a practical approach. , (2002).</li><li>Jiang, Y., Chen, J., Yen, K., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopically+Expressed+IL-34+Can+Efficiently+Induce+Macrophage+Migration+to+the+Liver+in+Zebrafish.">Ectopically Expressed IL-34 Can Efficiently Induce Macrophage Migration to the Liver in Zebrafish.</a> Zebrafish. 16 (2), 165-170 (2019).</li></ol>

The authors have nothing to disclose.

The protocol described here allows us to investigate the function of a chemokine on the behavior of macrophagein vivo and the procedure requires some technical expertise. In summary, there are several critical steps to avoid complications in the protocol: 1) select a suitable transgenic line which shows specific and strong transgenic signal to label the cell of interest; 2) select an appropriate tissue which is accessible for imaging and transgenic gene overexpression; 3) make a sensitive and specific RNA probe; 4) select an appropriate observation time window to accurately capture the cell behavior.
In the procedure of whole mount fluorescence in situ hybridization combined with immunostaining, the RNA probe used to detect gene expression should be sensitive and the signal need to be strong enough. In order to capture the gene function on cell behavior, a series of time points should be tested. For example, in observing the effect of il34 on macrophage migration, although the fabp10a promoter began to express at 2-3 dpf, macrophages accumulation in the liver was not obvious at that time. It is only by 4 dpf that the enrichment of macrophage in the liver becomes apparent. In addition, after in situ hybridization, the signal intensity of subsequent immunostaining will be affected. For example, comparing with GFP, DsRed is difficult to color in immunofluorescence staining after in situ hybridization, probably because of the different protein structures. Generally speaking, the signal intensity of immunostaining after whole mount fluorescence in situ hybridization would be less than that of single immunostaining.
In the live imaging step with the confocal microscope, it is necessary to keep the sample close to the bottom of the dish. When the embryos float in agarose, the working distance of the objective may be insufficient, also, the agarose between the objective and the sample would affect the quality of imaging. Besides, the number of samples for imaging at each time should be set properly. One must make sure that the time span between two scans of each fish would not be too long to lose the details of cell behavior. So this method is not suitable for tracking cells that move fast in thick tissues.
In conclusion, this protocol can be used to observe the function of chemokines on the behavior of a variety of cells such as macrophages, neutrophils, and T-cells. Here, we used IL-34, a recently identified ligand of CSF-1R function in chemotaxis6,7, as an ectopic expressed chemokine to induce macrophages migration. Most of the existing experimental models of cell chemotaxis are based on in vitro cell experiments, but the in vitro experiments sometimes are too simple to model the complex environment in vivo. Also, it is difficult to image the chemo-attraction abilityin vivo when simply look at the in vitro situation. This method utilized the specific advantages of zebrafish for direct cell behavior observation which is difficult for mice. The current method allowed us to quickly test the chemokine functions on cell behaviors within several days and make zebrafish a powerful model to study the molecular and cell biology.

Zebrafish is a small tropical hard-bones freshwater fish originated in India. Regarding the gene conservation, zebrafish have a similarity of 87% to the human1. It can provide us insights on related subjects of human by studying the gene regulation, protein function and cell behavior such as migration, proliferation et.al in zebrafish. Zebrafish embryo can be used to observe the development of early embryos at different stages after inhibiting pigment. Meanwhile, it takes only three months for zebrafish to develop into sexual maturity, then the zebrafish can produce hundreds of eggs every 4 days. Mini-size, simple breeding, strong reproductive capacity, these advantages make zebrafish culture very space-saving, conducive to large-scale culture. The traditional mammalian model mouse has a higher maintenance costs than zebrafish, therefore limiting the scale of mouse raising. In the aspect of early embryo development, mouse embryo is difficult to observe in live condition due to the characteristics of mouse embryo development in the mother womb. On the contrary, zebrafish embryos develop externally and are transparent, therefore they are easy to observe under a microscope. Furthermore, zebrafish is very easy to construct a variety of transgenic lines for related gene function research. Currently, various zebrafish transgenic lines are available to label different types of cells. It is very convenient now to construct transgenic lines to overexpress chemokines in specific locations and study the chemokines function on cell behavior in zebrafish.
Here, we provided a workflow to use zebrafish transgenic line to investigate the function of IL-34 on macrophage behavior in vivo2,3,4,5,6,7. Firstly, we constructed a liver-specific overexpression plasmid of the gene il34 and injected the plasmid into one-cell stage Tg (mpeg1: GFP) fish embryos which specifically labeled the macrophages by fluorescent protein GFP. Then, we used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the il34 expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. In these steps, we established and validated the cytokine-producing line and visually assessed the effects that can be seen on macrophage distribution. Finally, to investigate macrophage behavior in response to the cytokine, we used confocal live imaging to directly observe the macrophage migration to confirm the function of il34 on the macrophage migration in vivo.

<table><tbody><tr><td>&lt;strong&gt;Antibody&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa 488-Anti-Goat antibody</td><td>Invitrogen</td><td>A11055</td><td></td></tr><tr><td>Anti-Digoxigenin-HRP&amp;nbsp;</td><td>perkinelmer</td><td>NEF832001EA</td><td></td></tr><tr><td>Goat-Anti-GFP antibody</td><td>Abcam</td><td>ab6658</td><td></td></tr><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>21097</td><td></td></tr><tr><td>Cyanine 3 Plus Amplification Reagent</td><td>perkinelmer</td><td>NEL745001KT</td><td></td></tr><tr><td>E2 solution</td><td></td><td></td><td>15 mM&amp;nbsp; NaCl +0.5 mM KCl +1.0 mM MgSO&lt;sub&gt;4&lt;/sub&gt;+150 &amp;micro;M&amp;nbsp; KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; + 50 &amp;micro;M&amp;nbsp; Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; +1.0 mM CaCl&lt;sub&gt;2&lt;/sub&gt; + 0.7 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life</td><td>10099-133</td><td></td></tr><tr><td>Formamide</td><td>Diamond</td><td>A100314</td><td></td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Sigma</td><td>V900860</td><td></td></tr><tr><td>Heparin sodium</td><td>Sigma</td><td>H3149</td><td></td></tr><tr><td>Hybridization buffer(HB)</td><td></td><td></td><td>50% formamide+ 5&amp;times;SSC+9 mM sodium citrate+50 &amp;mu;g/ml heparin sodium+ 500 &amp;mu;g/mL tRNA+ 0.1% Tween20</td></tr><tr><td>KCl</td><td>Sigma</td><td>P5405</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P5655</td><td></td></tr><tr><td>Low melting agarose</td><td>Sigma</td><td>A9414</td><td></td></tr><tr><td>Methanol</td><td>GHTECH</td><td>1.17112.023</td><td></td></tr><tr><td>Methylene blue&amp;nbsp;</td><td>Sigma</td><td>M9140</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M2643</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S5136</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S5886</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>158127</td><td>Suspend 16 g of PFA in 400 ml of 1x PBS, heat at 60 &amp;deg;C&amp;nbsp; to dissolve about 30 min. This solution can be prepared in advance and stored at -4 &amp;deg;C. Caution. Manipulate with mask.</td></tr><tr><td>10&amp;times;PBS</td><td></td><td></td><td>14.2 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;+80 g NaCl+2 g KCl+ 2.4 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; in 1L ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Phenylthiourea (PTU)</td><td>Sigma</td><td>P7629</td><td></td></tr><tr><td>1&amp;times;Plus Amplification Diluent</td><td>perkinelmer</td><td>NEL745001KT</td><td></td></tr><tr><td>Proteinase K&amp;nbsp;</td><td>Fermentas</td><td>E00492</td><td></td></tr><tr><td>20&amp;times;Saline sodium citrate(SSC)</td><td></td><td></td><td>175.3 g NaCl+ 88.2 g sodium citrate in 1 L ddH&lt;sub&gt;2&lt;/sub&gt;O, PH 7.0</td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>A5040</td><td></td></tr><tr><td>Tricaine</td><td>Sigma</td><td>E10521</td><td></td></tr><tr><td>tRNA&amp;nbsp;</td><td>Sigma</td><td>R6625</td><td></td></tr><tr><td>Tween20</td><td>Sigma</td><td>P2287</td><td></td></tr><tr><td>&lt;strong&gt;Plasmid&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pBLK-fabp10a-il34-sv40</td><td></td><td></td><td>For &lt;em&gt;Tg (fab10a:il34)&lt;/em&gt; transgenic line generation</td></tr><tr><td>pBSK-il34</td><td></td><td></td><td>For &lt;em&gt;il34&lt;/em&gt; probe preparation</td></tr><tr><td>&lt;strong&gt;Fish&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Tg (mpeg1: GFP)&lt;/em&gt;</td><td></td><td></td><td>Label macrophages with GFP</td></tr><tr><td>&lt;em&gt;Tg (fabp10a: DsRed)&lt;/em&gt;</td><td></td><td></td><td>Label liver cells with DsRed</td></tr><tr><td>&lt;em&gt;Tg (fab10a:il34)&lt;/em&gt;</td><td></td><td></td><td>Over-expression IL-34 in liver cells</td></tr></tbody></table>

an ectopic chemokine expression model for testing macrophage recruitment in vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing to the transparent embryonic body of zebrafish, it is convenient for us to study the effect of one chemokine on the behavior of a certain type of cells in vivo. Here we provided a workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the chemokine expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish to study the function of IL-34 on macrophages in vivo.

The steps involved in the protocol of zebrafish are illustrated in Figure 2. First, we generated the pBLK-fabp10a-il34-sv40 construct in which il34 was driven by the fabp10a promoter (Figure 2). The construct was microinjected into one-cell stage Tg (mpeg1: GFP) zebrafish embryos which can label macrophages with GFP and WT embryos which were raised to adults to generate transgenic stable line (Figure 2). The expression of il34 was analyzed by whole mount fluorescence in situ hybridization (Figure 2 and Figure 3). Macrophages labeled by GFP were analyzed by immunostaining (Figure 2 and Figure 3). We used live imaging to directly observe whether macrophages would migrate into the liver under the il34 induction during 3-3.5 dpf (Figure 2, Figure 4, Supplementary Movie 1 and Supplementary Movie 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60161/60161fig1.jpg" /> 
Figure 1: Software operation of the confocal microscope live imaging. Open the ZEN black 2.3 software, install the living cell workbench onto the microscope carrier table, then click Locate (A) | Incubation | Temperature (B) to set the temperature to 29 &#176;C. Place the dish in the center of the living cell workbench, cover the fish with the E2 solution10 with tricaine. After all of these, click the Acquisition (C) menu, select the required scan mode and lasers in the Smart Setup (D) menu, then select Z-Stack and Position (E). Finally, click the Experiment Designer (F) menu, select the Enable Multi Block Experiment, in the first block, to find the sample under the low magnification, then switch to the high magnification, let the observed area in the center of the visual field, set the position and Z-Stack (G and H) information, select the appropriate laser intensity, scanning layers and imaging speed. Create a new block, and repeat the above steps. After setting up all blocks, set the appropriate number of loops (I) and start recording. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60161/60161fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60161/60161fig2.jpg" /> 
Figure 2: A workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific (liver) overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein (Tg: (mpeg1: GFP)). The injected WT embryos were raised to generate a stable transgenic line. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the gene expression and the number or location of macrophages of the transient injected embryos or stable line embryos (4 dpf). Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish (3-3.5 dpf) to study the function of IL-34 on macrophages in vivo. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60161/60161fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60161/60161fig3.jpg" /> 
Figure 3: Fluorescent WISH combine with immunostaining. This figure has been modified from Jiang et al.11. A total of 1.8 nL (30 ng/&#181;L) of the pBLK-fabp10a-il34-sv40 construct was microinjected into one-cell stage Tg (mpeg1: GFP) zebrafish embryos. (A) WISH of il34 expression (red) and whole-mount antibody staining of GFP expression (green) in 4 dpf embryo (6x). The whole body picture of the fish is made up of two separate images taken by confocal and stitched together in Photoshop. Insets are high magnification (20x) of the corresponding boxed regions (orange dotted areas). (B) Quantitative analysis of macrophage cell numbers in un-injected and construct injected embryos&#39; liver (shown in the white dotted area) and tail region (approximately between the 13th and 17th somite, shown between two white dotted lines). Data were analyzed by Mann Whitney U test, ** p &#60; 0.01 compared to control. n = 5, 5 for the 4 dpf injected and control fish. Bars: 200 &#181;m (white line); 50 &#181;m (yellow line). (C) WISH of il34 expression and whole-mount antibody staining of GFP expression in 4 dpf stable line embryo (6x). The whole body picture of the fish is made up of two separate images taken by confocal and stitched together in Photoshop. Insets are high magnification (20x) of the corresponding boxed regions (orange dotted areas). (D) Quantitative analysis of macrophage cell numbers in Tg (mpeg1: GFP) and Tg (fabp10a: il34; mpeg1: GFP) embryos&#39; liver (shown in the white dotted area) and tail region (approximately between the 13th and 17th somite, shown between two white dotted lines). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60161/60161fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60161/60161fig4.jpg" /> 
Figure 4: Confocal live imaging to directly observe macrophage behavior in the stable transgenic fish. This figure has been modified from Jiang et al.11. Micrographs of live imaging show the process of a macrophage (green, labeled by white arrows) passing by the liver (red) within 28 min in control fish (A) and the process of a macrophage (green, labeled by white arrows) migrating into the liver (red) within 28 min in IL-34 overexpressing fish (B). Scale bars = 40 &#181;m (white line). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60161/60161fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img src="/files/ftp_upload/60161/60161mov1v2.jpg" /> 
Supplementary Movie 1: Live imaging showing the process of macrophages (green, labeled by white arrows) migrating into the liver (red) within 2 h in IL-34 overexpressing fish. Scale bars =&#160;20 &#181;m (white line). This movie has been republished from Jiang et al.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60161/60161mov1.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img src="/files/ftp_upload/60161/60161mov2v2.jpg" /> 
Supplementary Movie 2: Live imaging showing the process of macrophages (green, labeled by white arrows) wandering around the liver (red) within 2 h in control fish. Scale bars = 20 &#181;m (white line). This movie has been republished from Jiang et al.11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60161/60161mov2.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo at JoVE.com

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing ...

an-ectopic-chemokine-expression-model-for-testing-macrophage

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

5.3K Views.  Southern Medical University. Het hier beschreven protocol stelt ons in staat om de functie van een chemokine en het gedrag van macrofaag in vivo te onderzoeken. De meeste bestaande experimentele modellen van cel chemotaxi's zijn gebaseerd op in vitro cel experimenten. Maar in vitro experimenten zijn soms te eenvoudig om de complexe omgeving in vivo te modelleren.Ook kunnen ze niet vertegenwoordigen de chemoattractie vermogen in vivo. Deze methoden maken gebruik van het specifieke voordeel van zebravissen voor dire...

Video: An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Cancer Research

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Immunology and Infection

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

Bioengineering

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 &#181;m/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

Here we describe methods using time-lapse, phase-contrast microscopy to image mouse resident peritoneal macrophages in a chemotactic complement C5a gradient. The protocols can be extended to other immune cells.

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a ...

Reprograming Model of Human Monocyte-derived Macrophages for In-vitro Assays

Cells of the monocyte-macrophage lineage are multifunctional and found in almost all body tissues. They coordinate innate and adaptive immunity&#180;s initiation and resolution phases, significantly affecting protective immunity and immune-mediated pathological injury. While tissue-resident macrophages are key players in maintaining homeostasis in a steady state, large amounts of monocytes are recruited from the peripheral blood into the tissue following damage or inflammatory insults. Monocyte-derived macrophages (M-DM) can differentiate into many dynamic subtypes, and their phenotypes and functions depend on the local tissue environment. 
To compare different stimuli or environmental conditions during M-DM differentiation and polarization, we standardized an in-vitro model of human nonpolarized M-DM M0 and some cardinal cytokine-polarized macrophages, IFN&#947;/LPS-derived M1, IL-4-derived M2a, and IL-10 or dexamethasone-derived M2c, to analyze skewing reprogramming of M-DM by flow cytometry and real-time PCR. We found that CD64, CD206, CD163, CD14, and MERTK can clearly discriminate unpolarized M0 and polarized M1, M2a, and M2c by flow cytometry. Moreover, we defined IRF1 and CXCL10 as specific genes for classical IFN&#947;/LPS-derived M1-, IRF4-, CCL22-, and TGM2-specific transcripts for IL-4-derived M2a, and the MERTK gene for dexamethasone-derived M2c. To summarize, our standardized M-DM protocol could give the cardinal in-vitro map to analyze the differentiation and polarization of human M-DM under diverse stimuli.

Monocytes and macrophages are very plastic and reprogrammable immune cells crucial for health and disease. They sense and respond to a broad range of stimuli by adopting specific differentiation programs and phenotypes. We have standardized an in-vitro model to study macrophage polarization and reprogramming, providing a valuable tool for research.

Cells of the monocyte-macrophage lineage are multifunctional and found in almost all body tissues. They coordinate innate and adaptive immunity&#180;s ...

Fluorescence Based Transwell Macrophage Attraction Assay: An In Vitro Method to Study Tumor-Macrophage Interaction Through Chemical-Induced Chemotaxis

Source: An, Z. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59907/in-vitro-assay-to-study-tumor-macrophage-interaction">In Vitro Assay to Study Tumor-macrophage Interaction.</a> J. Vis. Exp. (2019)
In this video, we study tumor-macrophage interaction using&#160;fluorescence-based transwell macrophage interaction assay. The assay involves the analysis of the migration of macrophages through chemical-induced chemotaxis.

Source: An, Z. et al. In Vitro Assay to Study Tumor-macrophage Interaction. J. Vis. Exp. (2019)
In this video, we study tumor-macrophage interaction ...

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

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