We thank Dr. Claire Davis and Dr. Veronique Miron (The Queen's Medical Research Institute, Edinburgh, United Kingdome) for initial help and discussions on the experimental approach and the QMRI Flow Cytometry and Cell Sorting Facility. This work was supported by a Cancer Research UK Career Establishment Award to Dr. Dirk Sieger.

1. Sample and Media Preparation
<ol>
	<li>Prepare embryo medium (E3) by dissolving 6.4 mM KCl, 0.22 mM NaCl, 0.33 mM CaCl2 2H2O, 0.33 mM MgSO4 7H2O in H2O.</li>
	<li>Collect zebrafish embryos immediately after fertilization (0 dpf).
	<ol>
		<li>Split zebrafish embryos into 50 per 90 mm petri dish. Raise embryos at 28.5 &#176;C in 50 mL of embryo medium (E3) treated with 200 &#181;M 1-phenyl 2-thiourea (PTU), from the end of the first day of development (0 dpf) for the duration of the experiment to inhibit pigmentation.</li>
		<li>Change the E3 + PTU medium daily for the duration of the experiment.</li>
		<li>Screen mpeg1:eGFP and NBT:DsRed larvae at 2 dpf using a florescent stereomicroscope for the positive transgene expression, GFP+ macrophages/microglia and DsRed+ Neurons.</li>
	</ol>
	</li>
	<li>Prepare all media the day before the experiment under a tissue culture hood to avoid contamination, then store them at 4 &#176;C.
	<ol>
		<li>Prepare media A by dissolving 15 mM Hepes and 25 mM D-Glucose in HBSS 1x.</li>
		<li>Prepare density gradient medium (100%) by mixing 9 volumes of the density gradient medium in 1 volume of HBSS 10x.</li>
		<li>Prepare the density gradient medium (22%) by diluting 22 mL of density gradient medium (100%) in 88 mL of DPBS 1x.</li>
		<li>Prepare DPBS 1x.</li>
		<li>Prepare E3 medium + Tricaine by mixing E3 medium with 450 &#956;M Tricaine.</li>
	</ol>
	</li>
</ol>
2. Homogenization
Note: All steps are performed 4 &#176;C.
<ol>
	<li>Add 1.5 mL Tricaine (15 mM) to 90 mm Petri dishes containing 50 larvae in 50 mL of E3 embryo medium treated with 200 &#181;M PTU to terminally anesthetize them.
	<ol>
		<li>Suck up 10 anesthetized larvae from the petri dishes with a 3 mL Pasteur plastic bulk pipette.</li>
		<li>Transfer anesthetized larvae 10 by 10 into a 55-mm petri dish filled with ice-cold E3 embryo medium + Tricaine.</li>
		<li>Under a stereomicroscope, align 10 larvae in the center of the petri dish. Then transect larval heads above the yolk-sac using surgical micro-scissors (exclude swim bladder to avoid floating heads).</li>
		<li>Suck up heads from the petri dishes with a 3 mL Pasteur plastic bulk pipette. Wait until all heads gather within the pipette tip and then transfer them into a glass homogenizer containing 1 mL ice-cold Media A (transfer in a minimal volume to reduce Media A dilution by E3 + Tricaine). Keep the glass homogenizer on ice. Use one homogenizer per experimental condition.</li>
		<li>Replace each small petri dish containing ice cold E3 + Tricaine with a new one every 30 min to assure that transection is performed in cold E3 + Tricaine medium.</li>
		<li>Replace the ice-cold Media A in the glass homogenizer when the color starts fading. 
		Note: Media A dilution can alter the head tissue due to temperature changes.</li>
		<li>Once all heads have been collected ( 600 heads/condition), remove the maximum volume of Media A from the glass homogenizer and replace it with 1 mL of fresh ice-cold Media A.</li>
		<li>Disrupt the brain tissue with a tight glass homogenizer on ice. Perform 40 rounds of crushing and turns for 3-5 dpf larvae and 50 for 7 and 8 dpf larvae.</li>
	</ol>
	</li>
	<li>Add 2 mL of Media A to cell suspension (1 mL Media A / 200 heads), that will dilute cells and reduce their agglomeration with myelin to facilitate their separation during the centrifugation in density gradient medium.
	<ol>
		<li>To eliminate cell agglomeration, run the cell suspension through a 40 &#181;m cell strainer placed on top of a cold 50 mL falcon tube maintained on ice. Repeat this operation 3 times.</li>
		<li>Transfer 1 mL of cell suspension into cold 1.5 mL tubes and spin them at 300 g for 10 min at 4 &#176;C.</li>
		<li>Remove supernatant using a 10-mL syringe + needle 23G x 1&#39;&#39;.</li>
	</ol>
	</li>
	<li>Re-suspend the cell pellet with 1 mL of ice-cold 22% density gradient medium gently overlaid by 0.5 mL of ice-cold DPBS 1x (do not mix them, an interphase between both solutions will be seen).
	<ol>
		<li>Spin tubes at 950 g without brake and slow acceleration for 30 min at 4 &#176;C. 
		Note: This step separates myelin from other cells by sequestering it at the interphase of DPBS 1x and 22% density gradient medium, whereas cells will pellet at the bottom of the tube. Myelin removal is more efficient when cell concentration is not too high.</li>
		<li>Using a 10-mL syringe + needle 23G x 1&#39;&#39; discard the maximum of DPBS, density gradient medium and myelin trapped at their interphase.</li>
		<li>Wash cells with 0.5 mL of Media A + 2% of normal goat serum (NGS), then spin tubes at 300 g for 10 min at 4 &#176;C.</li>
		<li>Discard the maximum of the supernatant, then pool all cell pellets from the same experimental condition together in 1 mL of Media A + 2% NGS.</li>
	</ol>
	</li>
	<li>If the cells of interest express a fluorescent protein like macrophages/microglia from mpeg1:eGFP or neurons from NBT:DsRed transgenic fish (screening of transgenic fish see 1.1.3), run the cell suspension through a 35 &#181;m cell strainer cap and transfer them into a cold 5 mL FACS tubes on ice, protected from light. 
	Note: Alternatively, immuno-staining of microglia can be performed.</li>
</ol>
3. Microglia Immuno-staining
Note: All steps are performed at 4 &#176;C.
<ol>
	<li>Re-suspend the cell pellet with 0.3 mL of Media A + 2% NGS. Split them in 3 x 1.5 mL tubes: one for unstained cells to measure auto-fluorescence from cells of interest, second for the secondary antibody (1/200) to measure the non-specific binding of the secondary antibody to microglia and third as a test (4C4 mouse monoclonal antibody (microglia specific) (1/20) + secondary antibody (1/200)).
	<ol>
		<li>Add Low Endotoxin, Azide-Free (LEAF) at 1% to cells (all tubes) to block CD16/CD32 interactions with the Fc domain of immunoglobulins. Incubate cells for 10 min with gentle agitation every 5 min.</li>
		<li>Add the 4C4 antibody (1/20) to cells (tube 3) and incubate for 30 min with gentle agitation every 10 min.</li>
		<li>Spin tubes at 300 g for 10 min at 4 &#176;C, then discard the supernatant.</li>
		<li>Wash once with 0.5 mL of Media A + 2% NGS, then spin tubes at 300 g for 10 min at 4 &#176;C.</li>
		<li>Re-suspend cell pellet with 0.5 ml of Media A + 2% NGS and incubate cells with LEAF at 1% for 10 min with gentle agitation every 5 min.</li>
		<li>Add secondary antibody (1/200) to cells (tube 2 and 3). Incubate cells for 30 min with gentle agitation every 10 min and light protection.</li>
		<li>Spin tubes at 300 g for 10 min at 4 &#176;C, then discard supernatant.</li>
		<li>Wash twice with 0.5 mL of Media A + 2% NGS, then re-suspend cell pellet with 1 mL of Media A + 2% NGS.</li>
	</ol>
	</li>
	<li>Run cell suspension through a 35 &#181;m cell strainer cap and transfer them into cold 5 mL FACS tubes on ice, protected from light.</li>
</ol>
4. Cell Sorting (FACS)
Note: Perform all steps at 4 &#176;C.
<ol>
	<li>Sort neurons, macrophages/microglia and microglia using a FACS. 
	Note: This step is usually performed by a staff member of the FACS facility and settings depend on the type of equipment used.
	<ol>
		<li>Add DAPI at a concentration of 1 &#181;g/mL in each FACS tube to label dead cells.</li>
		<li>Set up FACS and sort neurons, macrophages/microglia or microglia from all brain cells. Separate cells from debris in function of their size and granularity, then gate single-cells by forward scatter and side scatter. Exclude dead cells by DAPI labelling from live cells. Identify neurons, macrophages/microglia or microglia by their respective positive staining.</li>
	</ol>
	</li>
	<li>Collect cells in 1.5 mL tubes containing 1 mL of ice-cold Media A + 2% NGS on ice. Use different tubes for each cell type.
	<ol>
		<li>Spin tubes at 300 g for 10 min at 4 &#176;C and then discard the supernatant.</li>
		<li>Wash once with 0.5 mL of Media A then discard the maximum of supernatant.</li>
	</ol>
	</li>
</ol>
5. RNA Extraction
<ol>
	<li>Extract RNA from the different cell types separated by FACS. For RNA extraction use a specific kit and follow the manufacturer&#39;s guidelines. Ensure to work in an RNase free environment by cleaning workspace and pipettes with a RNase decontamination product and use filter tips.
	<ol>
		<li>Freshly prepare 1 mL of lysis buffer supplemented with 50 &#181;M &#946;-mercaptoethanol. 
		Note: Here, &#946;-mercaptoethanol is added to reduce RNA degradation.</li>
		<li>Prepare 70% and 80% ethanol solution using RNase free water.</li>
		<li>Lyse cells with 75 &#181;L of lysis buffer supplemented with 50 &#181;M &#946;-mercaptoethanol. Then use a shredder to improve cell disruption.</li>
		<li>Continue RNA extraction according to manufacturer&#39;s manual.</li>
		<li>At the end of the protocol, elute the RNA with 14 &#181;L of RNase free water to obtain a sufficient RNA concentration.</li>
	</ol>
	</li>
</ol>

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R., 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=Identification+of+a+conserved+and+acute+neurodegeneration-specific+microglial+transcriptome+in+the+zebrafish.">Identification of a conserved and acute neurodegeneration-specific microglial transcriptome in the zebrafish.</a> Glia. 65 (1), 138-149 (2016).</li><li>Hamilton, 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=A+Zebrafish+Live+Imaging+Model+Reveals+Differential+Responses+of+Microglia+Toward+Glioblastoma+Cells+In+Vivo.">A Zebrafish Live Imaging Model Reveals Differential Responses of Microglia Toward Glioblastoma Cells In Vivo.</a> Zebrafish. , (2016).</li><li>Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A., Lieschke, G. 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The authors have nothing to disclose.

The experimental protocol described here represents a robust and efficient method to isolate brain cells from zebrafish larvae from 3 to 8 dpf. Importantly, this is the first protocol that allows the specific isolation of microglia from larval zebrafish brains. The protocol is designed to preserve cell membrane integrity and to minimize potential modifications of gene expression occurring during the processing. This last point is crucial for the relevance of results based on the analysis of those isolated cellular genomic profiles. Indeed, microglia and macrophage polarization are strongly influenced by their microenvironment. At 37 &#176;C, these cells would have changed their gene expression profile in response to experimental conditions (injury (transection) response). Therefore, it was crucial to perform this experiment at 4 &#176;C prior to RNA extraction, to slow down cellular processes and metabolic activities. Furthermore, the mechanical brain tissue homogenization at 4 &#176;C was chosen instead of enzymatic tissue digestion at 37 &#176;C to avoid any impact on gene expression profiles.
It is important to highlight that this method is very quick; within a day several brain cell populations can be isolated from at least two different experimental conditions and their RNA extracted. The total length of the protocol depends on the number of larvae used for each condition as the transection of larval heads is the limiting step (&#8764; 350 heads/h). In general, to work with microglia from 3, 5 and 7 dpf it is recommended to transect &#8764; 600 heads per test to get enough RNA to extract (Table 1). As microglia represent the cell type with the lowest yield (approx. 112 cells per head at 7 dpf), the number of heads can be reduced for other cell types including macrophages (approx. 170 cells per head at 7 dpf).
Another advantage of this protocol is that once settings on the FACS sorter have been established, the same settings can be used for different experiments. It has been observed that cell populations fit perfectly from one experiment to another with gates previously designed, showing the reproducibility of experiments using this method.
A slight disadvantage of this method is the relatively low amount of RNA that is harvested. However, this limitation is more a biological issue than a technical issue, as the number of microglia is very low at early stages of brain development (Table 1). Because of this low quantity of RNA collected, amplification steps need to be considered to perform genome wide gene expression analysis. Fortunately, these amplification steps produce sufficient amounts of high quality cDNA. Thus, global changes in the gene expression profiles of isolated cells can be studied.
In conclusion, this protocol provides a robust method to isolate and study various CNS cell types from larval zebrafish brains. This can be applied to gain a deeper understanding of these cells during development as well as to study their role in disease.

Knowledge on brain development and brain diseases has significantly improved in the last decade since the first quantification of mouse brain transcriptomes1. Indeed, genome wide gene expression analysis gives us access to detailed genetic information on brain tissue and cells that can complement and improve observations made with other techniques and tools.
The zebrafish is a potent biological model, easy to breed and to modify genetically; its optical transparency at larval stages allows live imaging observations2. Unfortunately, compared to human and mouse, the number of available antibodies to perform immuno-staining is rather low. To remedy this, transgenic zebrafish fish lines are easily made by genetically modifying fish to express fluorescent proteins under cell type specific promoters. Transgenic zebrafish lines have been used in the past to study the role of macrophages and microglia during central nervous system (CNS) development and disease3,4,5,6. However, to gain a detailed understanding of these processes we need to understand changes in gene expression in the respective cell types. To this aim, we developed an experimental method to specifically isolate cells like neurons, macrophages and microglia from 3 to 8 days post-fertilization (dpf) larval zebrafish brains. For the establishment of the protocol, we worked with transgenic fish lines that express green fluorescent protein (GFP) in macrophages/microglia under the macrophage-expressed gene promoter (mpeg1:eGFP) and DsRed in neurons under the neural &#223;-tubulin promoter (NBT:DsRed)7,8,9. Furthermore, we performed immuno-staining of microglia using 4C4, a mouse monoclonal antibody that specifically stains zebrafish microglia10,11. Afterwards, ribonucleic acid (RNA) is extracted from these cells for further quantitative polymerase chain reaction (qPCR) or transcriptome analyses. This protocol has been designed to efficiently homogenize brain tissue from zebrafish larvae; collect neurons, macrophages/microglia and microglia without alteration of their plasma membrane integrity and finally extract RNA from these cells in high quality (RIN &#62; 7) and quantity to perform genomic analysis. Unlike previously published studies that use trypsin treatment at 37 &#176;C to digest brain tissue12,13, this protocol promotes work at 4 &#176;C till the RNA extraction step to reduce modifications of the gene expression profile. This step is crucial as microglia and macrophages are highly sensitive cells which respond to changes in their microenvironment immediately by altering their gene expression profile and polarization14,15,16.
The protocol, described here in detail, shows the isolation of neurons, macrophages and microglia from zebrafish larval brains, but virtually, it can be adapted to any other cell present within the brain - either by using transgenic fish lines or labelled with specific antibodies. This method will allow a better characterization of CNS cells through their genome wide gene expression analyses and will help to understand their role during development and brain diseases.

<table><tbody><tr><td>1-phenyl 2-thiourea (PTU)</td><td>Sigma</td><td>P7629</td><td></td></tr><tr><td>Hepes</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G8644-100ML</td><td></td></tr><tr><td>HBSS 1X</td><td>Gibco</td><td>14170-088</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-02</td><td></td></tr><tr><td>HBSS 10X</td><td>Gibco</td><td>14180-046</td><td></td></tr><tr><td>DPBS 1X</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Tricaine (MS222)</td><td>Sigma</td><td>A5040</td><td></td></tr><tr><td>Sterilin standard 90mm petri dishes</td><td>ThermoFisher</td><td>101VIRR</td><td></td></tr><tr><td>Surgical micro-scissors</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>3 mL Pasteur plastic bulk pipette</td><td>SLS</td><td>PIP4206</td><td></td></tr><tr><td>Glass homogenizer</td><td>Wheaton</td><td>357538</td><td></td></tr><tr><td>Sterilin standard 55mm petri dishes</td><td>ThermoFisher</td><td>P55V</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-02</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>50 ml polypropylen conical tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804 R</td><td></td></tr><tr><td>10 mL syringe</td><td>BD</td><td>302188</td><td></td></tr><tr><td>Needle 23G x 1&#039;&#039;</td><td>BD</td><td>300800</td><td></td></tr><tr><td>Normal goat serum (NGS)</td><td>Cell Signalling</td><td>5425S</td><td></td></tr><tr><td>35 &amp;mu;m cell strainer cap</td><td>BD</td><td>352235</td><td></td></tr><tr><td>FACS tubes</td><td>BD</td><td>352063</td><td></td></tr><tr><td>Low Endotoxin, Azide-Free (LEAF)</td><td>Biolegend</td><td>101321</td><td></td></tr><tr><td>Alexa Fluor 647 Goat Anti-Mouse IgG (H+L)</td><td>Life Technologies</td><td>A11008</td><td></td></tr><tr><td>Anti-4C4</td><td>Courtesy of Catherina Becker (University of Edinburgh)</td><td></td><td></td></tr><tr><td>FACS sorter FACSAria II</td><td>BD</td><td>QMRI, FACS facility</td><td></td></tr><tr><td>RNeasy Plus Micro Kit</td><td>QIAGEN</td><td>74034</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Gibco</td><td>&amp;nbsp;31350-010</td><td></td></tr><tr><td>QIAshredder</td><td>QIAGEN</td><td>79654</td><td></td></tr><tr><td>SsoAdvanced Universal SYBR Green Supermix</td><td>Bio-Rad</td><td>1725271</td><td></td></tr><tr><td>SuperScript III First-Strand Synthesis System</td><td>Invitrogen</td><td>18080-051</td><td></td></tr><tr><td>LightCycler 96 Real-Time PCR System&amp;nbsp;</td><td>Roche</td><td></td><td></td></tr><tr><td>Ovation RNA-Seq System V2</td><td>NuGEN</td><td>7102-32</td><td></td></tr><tr><td>Agilent RNA 6000 Pico reagents</td><td>Agilent</td><td>5067-1513</td><td></td></tr><tr><td>2100 Bioanalyzer</td><td>Agilent</td><td></td><td></td></tr><tr><td>RNaseZap</td><td>Ambion</td><td>AM9780</td><td></td></tr></tbody></table>

isolation and rna extraction of neurons, macrophages and microglia from larval zebrafish brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

The described protocol is a straightforward approach to isolate neurons, macrophages and microglia from zebrafish larval brains. From these isolated cells, significant amounts of high quality (RIN &#62; 7) RNA were extracted. The aim of this protocol is to isolate different types of cells from the CNS, with minimal modification of their gene expression profile to analyze and characterize cell properties and functions. Therefore, the entire protocol is performed at 4 &#176;C with a mechanical brain tissue homogenization. This method has been successfully used for two studies performed in the laboratory. In the first study, neurons and macrophages/microglia were isolated from 8 dpf mpeg1:eGFP+/NBT:DsRed+ larvae (Figure 1). FACS allowed cell separation from debris in function of their size (FSC-A) and granularity (SSC-A) (Figure 1A). Single cells were then separated from doublets or cell agglomerates (Figure 1B). From the single cell population, a gate was drawn to eliminate dead cells (DAPI+). The corresponding dot plot revealed that this experimental protocol preserves cell plasma membrane integrity, as the rate of dead cells is only 26.7% (Figure 1C). Finally, neurons (DsRed+) and macrophages/microglia (GFP+) were easily segregated from the live cell population gates. The neuron population (23.1 %) appeared to be more prominent than the macrophages/microglia population (1.56 %) within the brain (Figure 1D). This protocol has allowed to isolate RNA from those cells to perform subsequent qPCR analyses to compare the expression of specific genes between neurons and macrophages/microglia. Figure 2 shows neuronal and macrophages/microglia gene expression levels of proliferating cell nuclear antigen (pcna)against the &#946;-actin house-keeping gene as an example.
For the second study this method focused on microglia isolation from 3, 5 and 7 dpf larval brains. In contrast to the experiment described above, cells were isolated by immuno-staining using 4C4, an antibody which specifically labels microglia (Figure 3 A-D). As previously described, microglia (4C4+) were selected from live cells and collected (Figure 3D). Microglia numbers within zebrafish larval brains are variable (Table 1), and very low at 3 dpf (&#8764; 25 per fish). Quality and quantity of extracted RNA from those cells were measured using a micro-capillary electrophoresis based system. Results obtained of extracted RNA from microglia of 5 dpf larval zebrafish brains have been provided to illustrate an example of RNA analysis (Table 1 (5 dpf; experiment 4)). Figure 4 shows the electrophoresis trace and its graphic representation obtained for this sample with a clear visualization of ribosomal RNA (28s and 18s). This data is necessary to calculate sample RIN and to determine RNA concentration. Table 1 summarizes the number of isolated microglia per fish, the amount of RNA per microglia and the RIN score obtained for each different experiment at 3, 5 and 7 dpf. The amount and the quality of extracted RNA from isolated microglia using this method allowed us to amplify the RNA into cDNA using a kit. Quality and quantity tests provided by Edinburgh Genomics confirm that the amplified cDNA is of sufficient quality for library preparation and subsequent sequencing. Figure 5 shows the size distribution of cDNA fragments and their amount measured using an electrophoretic system. In this sample, the cDNA had a mean size of 299bp at a concentration of 36100 pmol/l. Table 2 illustrates respectively quality and quantity tests made on amplified cDNA from RNA samples (Table 1 (5 dpf; experiment 4)). The amplified cDNA has been used successfully for sequencing.
Several studies performed in the laboratory confirmed that the quality and quantity of extracted RNA from neurons, macrophages and microglia can be used for subsequent qPCR and genome wide gene expression analyses. Therefore, this experimental protocol can be used to reliably isolate different types of CNS cells without altering their membrane integrity and limiting modification of their gene expression profile.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57431/57431fig1.jpg" /> 
Figure 1: FACS sorting for neurons and macrophages/microglia from mpeg1:GFP+/NBT:DsRed+ 8 dpf zebrafish larvae. (A-C) Successive gating shows sequential selection of all brain cells (A), single cells by forward scatter and side scatter (B). (C) Dead cells were excluded by DAPI labelling. (D) Neurons and macrophages/microglia were identified respectively by DsRed and GFP positive staining. <a href="https://cloudflare.jove.com/files/ftp_upload/57431/57431fig1large.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/57431/57431fig2.jpg" /> 
Figure 2: Gene expression analysis for pcna and &#946;-actin in neurons and macrophages/microglia. RNA from isolated neurons and macrophages/microglia can be transcribed into cDNA for use in quantitative PCR analysis. mRNA expression levels of pcna against &#946;-actin house-keeping gene in isolated neurons and macrophages/microglia determined by qPCR (N = 3). Fold change was measured using the comparative (&#916;&#916;CT) method. Error bar represent mean &#177; SEM. <a href="https://cloudflare.jove.com/files/ftp_upload/57431/57431fig2large.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/57431/57431fig3.jpg" /> 
Figure 3: FACS sorting for microglia from 3 dpf zebrafish larvae. (A-C) Successive gating show sequential selection of all brain cells (A), single cells by forward scatter and side scatter (B). (C) Dead cells were excluded by DAPI labelling. (D) Microglia were identified by 4C4 positive staining. <a href="https://cloudflare.jove.com/files/ftp_upload/57431/57431fig3large.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/57431/57431fig4.jpg" /> 
Figure 4: Micro-capillary electrophoresis results of extracted RNA from 5 dpf zebrafish microglia. The two tall peaks are the 18S and 28S ribosomal RNA. RNA integrity number (RIN) was automatically calculated by the bioanalyzer software using the generated ratio of the 18S and 28S ribosomal subunits and the analysis of the entire electrophoretic trace. Microglia RNA has a RIN 8.6. <a href="https://cloudflare.jove.com/files/ftp_upload/57431/57431fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57431/57431fig5.jpg" /> 
Figure 5: Quality and quantity tests of amplified cDNA from RNA sample of 5 dpf zebrafish microglia. The image shows the cDNA fragment size distribution of the analyzed sample with a mean size of 299 bp. <a href="https://cloudflare.jove.com/files/ftp_upload/57431/57431fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Condition</td>
			<td>Experiment</td>
			<td>Fish number</td>
			<td>Cell number</td>
			<td>Cell number per fish</td>
			<td>RNA concentration (pg/ul)</td>
			<td>Total RNA&#160; (pg)</td>
			<td>RNA amount per cell (pg)</td>
			<td>RIN score</td>
		</tr>
		<tr>
			<td>3dpf</td>
			<td>1</td>
			<td>700</td>
			<td>11922</td>
			<td>17.03</td>
			<td>126</td>
			<td>1512</td>
			<td>0.13</td>
			<td>8</td>
		</tr>
		<tr>
			<td>3dpf</td>
			<td>2</td>
			<td>600</td>
			<td>22527</td>
			<td>37.55</td>
			<td>253</td>
			<td>3036</td>
			<td>0.13</td>
			<td>7.9</td>
		</tr>
		<tr>
			<td>3dpf</td>
			<td>3</td>
			<td>600</td>
			<td>18688</td>
			<td>31.15</td>
			<td>255</td>
			<td>3060</td>
			<td>0.16</td>
			<td>7.7</td>
		</tr>
		<tr>
			<td>3dpf</td>
			<td>4</td>
			<td>600</td>
			<td>11121</td>
			<td>18.54</td>
			<td>189</td>
			<td>2268</td>
			<td>0.20</td>
			<td>7.8</td>
		</tr>
		<tr>
			<td>3dpf</td>
			<td>5</td>
			<td>600</td>
			<td>15581</td>
			<td>25.97</td>
			<td>131</td>
			<td>1572</td>
			<td>0.10</td>
			<td>8.4</td>
		</tr>
		<tr>
			<td>3dpf</td>
			<td>6</td>
			<td>600</td>
			<td>11965</td>
			<td>19.94</td>
			<td>256</td>
			<td>3072</td>
			<td>0.26</td>
			<td>8.2</td>
		</tr>
		<tr>
			<td>&#160;5dpf</td>
			<td>1</td>
			<td>600</td>
			<td>58629</td>
			<td>97.72</td>
			<td>362</td>
			<td>4344</td>
			<td>0.07</td>
			<td>7.4</td>
		</tr>
		<tr>
			<td>&#160;5dpf</td>
			<td>2</td>
			<td>600</td>
			<td>32510</td>
			<td>54.18</td>
			<td>348</td>
			<td>4176</td>
			<td>0.13</td>
			<td>8.1</td>
		</tr>
		<tr>
			<td>&#160;5dpf</td>
			<td>3</td>
			<td>600</td>
			<td>77884</td>
			<td>129.81</td>
			<td>594</td>
			<td>7128</td>
			<td>0.09</td>
			<td>8.3</td>
		</tr>
		<tr>
			<td>&#160;5dpf</td>
			<td>4</td>
			<td>600</td>
			<td>50755</td>
			<td>84.59</td>
			<td>305</td>
			<td>3660</td>
			<td>0.07</td>
			<td>8.6</td>
		</tr>
		<tr>
			<td>&#160;5dpf</td>
			<td>5</td>
			<td>600</td>
			<td>44967</td>
			<td>74.95</td>
			<td>134</td>
			<td>1608</td>
			<td>0.04</td>
			<td>7.6</td>
		</tr>
		<tr>
			<td>&#160;5dpf</td>
			<td>6</td>
			<td>600</td>
			<td>51031</td>
			<td>85.05</td>
			<td>163</td>
			<td>1956</td>
			<td>0.04</td>
			<td>7.9</td>
		</tr>
		<tr>
			<td>7dpf</td>
			<td>1</td>
			<td>600</td>
			<td>60496</td>
			<td>100.83</td>
			<td>183</td>
			<td>2196</td>
			<td>0.04</td>
			<td>7.6</td>
		</tr>
		<tr>
			<td>7dpf</td>
			<td>2</td>
			<td>450</td>
			<td>55517</td>
			<td>123.37</td>
			<td>183</td>
			<td>2196</td>
			<td>0.04</td>
			<td>7.8</td>
		</tr>
		<tr>
			<td>7dpf</td>
			<td>3</td>
			<td>600</td>
			<td>88897</td>
			<td>148.16</td>
			<td>465</td>
			<td>5580</td>
			<td>0.06</td>
			<td>8.1</td>
		</tr>
		<tr>
			<td>7dpf</td>
			<td>4</td>
			<td>600</td>
			<td>63008</td>
			<td>105.01</td>
			<td>356</td>
			<td>4272</td>
			<td>0.07</td>
			<td>8.4</td>
		</tr>
		<tr>
			<td>7dpf</td>
			<td>5</td>
			<td>350</td>
			<td>34956</td>
			<td>99.87</td>
			<td>245</td>
			<td>2940</td>
			<td>0.08</td>
			<td>8.1</td>
		</tr>
		<tr>
			<td>7dpf</td>
			<td>6</td>
			<td>600</td>
			<td>63887</td>
			<td>106.48</td>
			<td>341</td>
			<td>4092</td>
			<td>0.06</td>
			<td>7.8</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of microglia isolation and RNA extraction data from 3, 5 and 7 dpf zebrafish larvae. 
<table fo:font-size="7" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Internal Sample ID</td>
			<td>External Sample ID</td>
			<td>Qubit (ng/ul)</td>
			<td>Qubit(ng/ul)</td>
			<td>Average concentration (ng/ul)</td>
			<td>Volume (ul)</td>
			<td>ug received</td>
			<td>Pass/fail for minimum&#160; concentration</td>
			<td>Pass/fail for minimim quantity</td>
			<td>Pass/fail for recommended quantity</td>
		</tr>
		<tr>
			<td>10907SD0010</td>
			<td>5 dpf&#160; (experiment 4)</td>
			<td>58.8</td>
			<td>58.4</td>
			<td>58.6</td>
			<td>30</td>
			<td>1.76</td>
			<td>Pass</td>
			<td>Pass</td>
			<td>Pass</td>
		</tr>
		<tr>
			<td colspan="10">Sample Requirements for Library Prep:</td>
		</tr>
		<tr>
			<td>Library Prep</td>
			<td>Minimum Quantity (ng)</td>
			<td>Recommended Quantity (ng)</td>
			<td>Minimum Concentration ng/uL</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TruSeq Nano gel free 350 bp insert library from cDNA</td>
			<td>600</td>
			<td>1100</td>
			<td>10</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Quantity tests of amplified cDNA from RNA sample of 5 dpf zebrafish microglia.

Watch this Scientific Journal Video about Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains at JoVE.com

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

isolation-rna-extraction-neurons-macrophages-microglia-from-larval

Research

JoVE Journal

Neuroscience

13.6K Views.  University of Edinburgh. We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

Video: Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Developmental Biology

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Biology

Processing Gut Cells from Zebrafish Larvae for Single-Cell RNA Sequencing

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

Author Spotlight: Isolating and Analyzing Intestinal Cells of Zebrafish Larvae for Investigating Transcriptomic Aspects of Gastrointestinal Development 

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Brain Cell Isolation from Zebrafish Larvae: A Technique to Obtain Intact Neurons, Macrophages, and Microglia from Zebrafish Brain Tissue

Source: Mazzolini, J. et al. <a href="https://www.jove.com/v/57431/isolation-rna-extraction-neurons-macrophages-microglia-from-larval">Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains.</a> J. Vis. Exp. (2018)
This video describes a protocol to isolate neurons, macrophages, and microglia from larval zebrafish brains by mechanically disrupting the brain tissue. The purified cell populations can further be used for genomic analysis.

Source: Mazzolini, J. et al. Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains. J. Vis. Exp. (2018)
This ...

JoVE Encyclopedia of Experiments

Cancer Research

Cancers of the Nervous System

Ex vivo studies

Microglia Isolation by Immunostaining Coupled Cell Sorting: An Immunostaining Method to Isolate Microglia from Zebrafish Brain Cells Using Fluorescent Activated Cell Sorting

Source: Mazzolini, J. et al. <a href="https://www.jove.com/v/57431/isolation-rna-extraction-neurons-macrophages-microglia-from-larval">Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains.</a> J. Vis. Exp. (2018)
In this video, we isolate zebrafish microglial cells via immunostaining-coupled cell sorting. The isolated microglia can be used in downstream experiments to understand their role in brain pathologies.

Source: Mazzolini, J. et al. Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains. J. Vis. Exp. (2018)
In this ...