XL was supported in part by the National Key R&#38;D Program of China (2017YFE0196600), and the National Natural Science Foundation of China (Grant Nos. 31771395, 31571518). Q.S. was supported by the National Key Research and Development Program of China (2017YFC1001703) and the Key Research and Development Program of Zhejiang Province (2017C03009). W.X. was supported by the Natural Science Foundation of Zhejiang province (LY18H020002) and Science Technology Department of Zhejiang Province (2017C37057).

All the animal procedures have been approved by the Animal Ethics Committee of Zhejiang University.
1. The Culture of Adult Neural Stem Cells and Neurons
<ol>
	<li>Isolate adult neural stem cells from the forebrain of an adult (8-10 week old) C57/BL6 male mouse as described previously31,32.</li>
	<li>Culture adult neural stem cells in DMEM/F-12 medium containing 20 ng/mL FGF-2, 20 ng/mL EGF, 2% B27 supplement, 1% antibiotic-antimycot...

<ol>
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Epigenetic modifications play essential roles during brain development, maturation, and function. As a stable marker for DNA modification, dynamic 5-hmC responds to behavioral adaptation, neuronal activity, and is positively correlated with gene expression; thus, it is involved in the normal function of the brain and neurological disorder4. To explore its function in cells and tissues, it is necessary to detect the existence of 5-hmC and compare the level before and after treatment. Here, we demon...

Epigenetic modifications, including DNA modification, histone modification and RNA modification, have been shown to play essential functions in diverse biological processes and diseases1,2,3,4,5,6,7. For a long time, DNA methylation (i.e., 5-methylcytosine (5-mC)) has been viewed as a highly stable epigenetic marker and can not be further modified in the genome. Recently, it has been found that 5-mC could be oxidized to 5-hydroxymethylcytosine(5-hmC) by TET (Ten-eleven translocations) family proteins including TET1, TET2, and TET38,9. Further studies show that 5-hmC could serve as a stable marker and play biological roles through regulating gene expression4,10,11,12.
The present evidences indicate&#160;that 5-hmC is highly enriched in neuronal tissues/cells relative to other types of tissues in mammals, and exhibits dynamic features during neuronal development13,14. In the&#160;neuronal system, 5-hmC mediated epigenetic modifications play an important role in regulating neural stem cells, neuronal activity, learning and memory, and is involved in multiple neurological disorders including Rett syndrome, autism, Alzheimer&#8217;s disease, Huntington&#8217;s disease, etc.2,13,15,16,17,18,19,20.
There are several approaches for detecting 5-hmC in cells and tissues14,21,22,23,24. Here, we describe two methods to detect the existence of 5-hmC and quantify the global level of 5-hmC: immunofluorescence staining and DNA dot-blot. These two methods are convenient and sensitive, and have been successfully used in previous studies25,26,27,28,29,30. The key steps of these two methods are DNA denaturation. For immunofluorescence staining of 5-hmC, pre-treatment of samples with 1 M HCl is required. For 5-hmC dot-blot, DNA denaturation is performed with&#160;NaOH solution. These two methods together with next-generation sequencing are very useful tools for investigating the function of 5-hmC.

<table>
	<tbody>
		<tr>
			<td>4&#39;-6-diamidino-2-phenylindole (DAPI )</td>
			<td>Sigma-Aldrich</td>
			<td>D8417</td>
		</tr>
		<tr>
			<td>Adobe Photoshop software</td>
			<td>Adobe Inc.</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-rabbit IgG</td>
			<td>Thermo Fisher</td>
			<td>A11008</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 goat anti-mouse IgG</td>
			<td>Thermo Fisher</td>
			<td>A11001</td>
		</tr>
		<tr>
			<td>anti-5-hydroxymethylcytosine</td>
			<td>Active Motif</td>
			<td>39769</td>
		</tr>
		<tr>
			<td>anti-NeuN</td>
			<td>Millipore</td>
			<td>MAB377</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>12587-010</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>12580-010</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
		</tr>
		<tr>
			<td>Cryostat microtome</td>
			<td>Leica</td>
			<td>CM1950</td>
		</tr>
		<tr>
			<td>DMEM/F-12 medium</td>
			<td>OmegaScientific</td>
			<td>DM25</td>
		</tr>
		<tr>
			<td>epidermal growth factor</td>
			<td>PeproTech</td>
			<td>100-15</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor-basic</td>
			<td>PeproTech</td>
			<td>100-18B</td>
		</tr>
		<tr>
			<td>forskolin</td>
			<td>Sigma-Aldrich</td>
			<td>F6886</td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Thermo</td>
			<td>35050061</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-149</td>
		</tr>
		<tr>
			<td>neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>Vector Laboratories</td>
			<td>Z0325</td>
		</tr>
		<tr>
			<td>nylon membrane (Hybond&#8482;-N+ )</td>
			<td>Amersham Biosciences</td>
			<td>RPN303B</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Leica</td>
			<td>14020108926</td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>phenol: chloroform: isoamyl alcohol (25: 24:1 )</td>
			<td>Sigma-Aldrich</td>
			<td>516726</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>P0899-10</td>
		</tr>
		<tr>
			<td>proteinase K</td>
			<td>VVR</td>
			<td>39450-01-6</td>
		</tr>
		<tr>
			<td>retinoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Solarbio</td>
			<td>T8210</td>
		</tr>
	</tbody>
</table>

the detection of 5-hydroxymethylcytosine in neural stem cells and brains of mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

To reveal the distribution of 5-hmC in the hippocampus of adult mice, we performed immunofluorescence with antibodies against neuronal cells (NeuN) and 5-hmC. In the hippocampus, 5-hmC co-localized well with neuronal cell marker NeuN (Figure 1A-H), suggesting an enrichment of 5-hmC in neurons.
To determine the dynamics of 5-hmC during neuronal development, a dot-blot was first performed with DNA samples isolated from proliferating and differentiat...

Watch this Scientific Journal Video about The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice at JoVE.com

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Here, we present a protocol to detect 5-hydroxymethylcytosin in cells and the brain tissues using immunofluorescence, the new method, and the DNA dot-blot. 5-hydroxymethylcytosin, i.e. 5hmC, mitigated epigenetic modification, plays important function in regulating gene expression and involved in multiple neurological disorders.Actually, our methods can be used to detect 5hmC in multiple cells and the tissues. It's convenient and can be performed with common equipments in the lab. Begin by positioning the mouse face up on a plastic board and fixing each limb with sticky tape.Use surgical scissors to cut through the skin and muscle, and open the thoracic cavity. Expose the heart and cut off a small part of the right atrium with fine surgical scissors. Using a 10-milliliter disposable, sterilized syringe, perfuse the mouse from the left ventricle with cold PBS.Then, perfuse the mouse with 4%PFA until it is stiff. Then, open the skull with bone forceps and remove the brain. Place the brain into a 15-milliliter centrifuge tube filled with five milliliters of 4%PFA and store it at four degrees Celsius.After 24 hours, transfer the brain into a 30%sucrose solution and leave it at four degrees Celsius for complete dehydration. Prior to sectioning, imbed the brain in optimal cutting temperature compound and cool to minus 20 degrees Celsius for at least one hour. Section brain samples at a thickness of 20 to 40 micrometers using a cryostat microtome.Collect sections into PBS and store at four degrees Celsius. Pick up the brain sections of interest and put them into a 24-well plate with PBS. Wash sections with PBS on a shaker for 10 minutes.Remove the PBS and treat with preheated one-molar hydrochloric acid at 37 degrees Celsius for 30 minutes. Hydrochloric acid is corrosive and please prepare it in the chemical hold. Then, wash the samples three times with PBS for five minutes and block them with PBS containing 3%normal goat serum and 0.1%Triton X-100.Leave the samples on a shaker at room temperature for one hour. Add specific primary antibodies to the sections and incubate overnight at four degrees Celsius on a shaker. The next day, take the samples out and leave them at room temperature for one hour.Wash the samples three times with PBS and add secondary antibodies. Cover the plate with aluminum and leave it at room temperature for one hour while shaking. After the incubation, wash the samples three times with PBS and mount them onto slides.Add 100 to 150 microliters of anti-fade mounting medium, cover with a premium cover glass, and seal with nail polish. Image the brain sections with a regular or confocal microscope. To isolate genomic DNA, start by dissecting the hippocampus, cortex, and cerebellum tissues on an ice-cooled dish.Add one milliliter of DNA lysis buffer and grind the tissues. Transfer the sample into a clean microcentrifuge tube and add 250 micrograms of proteinase-K for every 600 microliters of lysis buffer. Then, add about 50 micrograms of RNase A per sample and incubate for at least 12 hours at 37 degrees Celsius.After the incubation, add an equal volume of phenol chloroform isoamyl alcohol and mix thoroughly. Centrifuge the mixture according to manuscript directions and transfer the supernatant to a new tube. Add 600 microliters of chloroform to the supernatant, mix thoroughly, and centrifuge according to manuscript directions.Transfer the supernatant to a new tube and add 500 microliters of isopropanol. Mix thoroughly and centrifuge again to precipitate the DNA. After the centrifugation, discard the supernatant, and wash the DNA pellet with 70%ethanol according to manuscript directions.Air dry the DNA pellet completely and then, dissolve it in Tris-HCl buffer. Prior to starting, prepare the required solutions and sample mixture according to manuscript directions. Denature the DNA sample by heating it to 100 degrees Celsius for 10 minutes and then, place it on ice.Cut the appropriate size of a nylon membrane and rinse it with 6X SSC. Place the membrane on a dot-blot apparatus and connect the vacuum pump. Spot six-microliter dots onto the membrane and hybridize for 30 minutes at 80 degrees Celsius.Then, block the sample membrane with fat-free milk and Tris-buffered saline, or TBS, for one hour. Add the polyclonal rabbit anti-5-hydroxymethylcytosin antibody to the membrane and incubate overnight at four degrees Celsius. The next day, leave the sample membrane at room temperature for one hour, then wash the membrane three times with TBS.Incubate the membrane with the anti-rabbit secondary antibody for 30 minutes and then, wash three times with TBS. Visualize the chemiluminescence signals and quantify their intensity. This protocol can be used to visualize 5-hydroxymethylcytosin, or 5hmC, in the hippocampus of adult mice.Immunofluorescence with antibodies against neuronal cells, the 5hmC reveals that there is an enrichment of 5hmC in neurons. Dynamics of 5hmC during neuronal development can be determined with a dot-blot assay of DNA samples isolated from proliferating and differentiated adult neural stem cells. Global levels of 5hmC significantly increase during differentiation and the level of 5hmC in neurons is higher than in neural stem cells.The key step of this method is to make sure that complete denaturation of DNA samples.

the-detection-5-hydroxymethylcytosine-neural-stem-cells-brains

Research

JoVE Journal

Neuroscience

6.3K visualizações.  Zhejiang University. Aqui, apresentamos um protocolo para detectar 5-hidroximetilcytosina nas células e nos tecidos cerebrais usando imunofluorescência, o novo método, e o dna ponto-mancha. 5-hidroxietilcytosina, ou seja, 5hmC, modificação epigenética atenuada, desempenha importante função na regulação da expressão genética e envolvida em múltiplos distúrbios neurológicos.Na verdade, nossos métodos podem ser usados para detectar 5hmC em múltiplas células e tecidos. É conveniente e pode s...