Name: immunofluorescence to monitor the cellular uptake of human lactoferrin and its associated antiviral activity against the hepatitis c virus
Uploaded: 2015-10-01T13:30:00
Description: Watch this Scientific Journal Video about Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus at JoVE.com

This work was funded by both the&#160;Canadian Institutes of Health Research&#160;and&#160;Natural Sciences and the Engineering Research Council of Canada. M. Bisaillon is a Chercheur Boursier Senior from the Fonds de Recherche en Sant&#233; du Qu&#233;bec and also a member of the Centre de Recherche Clinique du Centre Hospitalier Universitaire de Sherbrooke. We thank Dr. Ralf Bartenschlager for the generous gift of the HCV replicon system. We also thank Dr. Charles Rice and Dr. Daniel Lamarre for kindly providing the hepatic cell line. We also want to thank Guillaume Tremblay for technical assistance.

1.细胞制备与处理<ol><li>在一个24孔板，把一个玻璃盖在孔的底部。用磷酸盐缓冲盐水（PBS）洗涤各孔。 </li><li>种子Huh-7细胞在每孔配套的HCV亚基因组复制到5×10 4个细胞的密度/孔。如果没有治疗做，细胞可以在更高的密度接种。培养基是Dulbecco氏改良的Eagle培养基（DMEM），补充有10％胎牛血清（FBS），2mM L-谷氨酰胺，1mM丙酮酸钠和250毫克/毫升的G-418（以维持复制子）。 </li><li>在37℃和5％?...

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	<li>Odell, I. D., Cook, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+techniques.">Immunofluorescence techniques.</a> J Invest Derm. 133 (1), e4 (2013).</li><li>Portugal, J., Waring, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assignment+of+DNA+binding+sites+for+4&#8217;,6-diamidine-2-phenylindole+and+bisbenzimide+(Hoechst+33258).+A+comparative+footprinting+study.">Assignment of DNA binding sites for 4&#8217;,6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study.</a> Biochim Biophys Acta. 949 (2), 158-168 (1988).</li><li>Lichtman, J. W., Conchello, J. -. A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy.">Fluorescence microscopy.</a> Nat Methods. 2 (12), 910-919 (2005).</li><li>St Croix, C. M., Shand, S. H., Watkins, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy:+comparisons,+applications,+and+problems.">Confocal microscopy: comparisons, applications, and problems.</a> BioTechniques. 39, S2-S5 (2005).</li><li>Yi, M., Kaneko, S., Yu, D. Y., Murakami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+envelope+proteins+bind+lactoferrin.">Hepatitis C virus envelope proteins bind lactoferrin.</a> Journal of virology. 71, 5997-6002 (1997).</li><li>Wakabayashi, H., Oda, H., Yamauchi, K., Abe, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactoferrin+for+prevention+of+common+viral+infections.">Lactoferrin for prevention of common viral infections.</a> J Inf Chem. 20 (11), 666-671 (2014).</li><li>Picard-Jean, F., Bouchard, S., Lariv&#233;e, G., Bisaillon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+inhibition+of+HCV+replication+represents+a+novel+mechanism+of+action+by+the+innate+immune+Lactoferrin+protein.">The intracellular inhibition of HCV replication represents a novel mechanism of action by the innate immune Lactoferrin protein.</a> Antiviral research. 111, 13-22 (2014).</li><li>Johnson, G. D., Davidson, R. S., McNamee, K. C., Russell, G., Goodwin, D., Holborow, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fading+of+immunofluorescence+during+microscopy:+a+study+of+the+phenomenon+and+its+remedy.">Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy.</a> J Immunol Methods. 55 (2), 231-242 (1982).</li><li>Green Remington, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein:+a+perspective.">fluorescent protein: a perspective.</a> Protein Sci. 20 (9), 1509-1519 (2011).</li></ol>

丙型肝炎病毒的流行仍然是一个全球性的威胁，有80％的新感染者发展为慢性感染的，这使他们肝硬化，肝衰竭或肝癌的危险。直接作用的靶向HCV复制和成熟抗病毒剂代表素抗HCV药物，如最近由两个NS3 N-末端蛋白酶抑制剂（用boceprevir和替拉瑞韦）的监管机构批准证明。所述的hLF抗HCV活性的目前认为表现为病毒进入抑制剂，结合循环病毒粒子和阻止它们进入到他们的肝细胞靶。我们对铁蛋白的研究?...

Immunofluorescence is a technique that uses a fluorescence microscope to visualize the distribution and/or localization of a target molecule in a biological sample. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell1. It is typically used on tissue sections and cultured cell lines in order to analyze the distribution/localization of various biological molecules such as proteins, nucleic acids and glycans. It should be noted that immunofluorescence is often used in combination with other non-antibody methods of fluorescent staining such as the 4',6-diamidino-2-phenylindole (DAPI) stains which are typically used to label DNA2. Moreover, this technique involves fixation of the cells which allows the analysis of cells at a specific time.
Different types of microscopes can be used to analyze immunofluorescence samples. The simplest is the epifluorescence microscope (Figure 1) for which excitation of the fluorochrome and detection of the fluorescence are done through the same light path3. Because most of the excitation light is transmitted through the sample, only reflected excitatory light can reach the objective together with the emitted light. This approach unfortunately leadsto a frequent high signal to noise ratio.In contrast, confocal microscopy (Figure 2) offers a distinct advantage for increasing optical resolution and contrast by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light4. This approach allows the reconstruction of three-dimensional structures from the obtained images. However, since an important fraction of the light from the sample is blocked at the pinhole, long exposures are often required.
There are two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect). Direct immunofluorescence involves a primary antibody linked with a fluorochrome (Figure 3). This method is less frequently used because it provides lower signal, involves higher cost and less flexibility1. Moreover, such antibodies are generally harder to find commercially. On the other hand, the direct attachment of the fluorochrome to the antibody significantly reduces the number of steps in the procedure, saving time and frequently reducing non-specific background signal. This also limits the possibility of antibody cross-reactivty.
Indirect immunofluorescence involves a primary unlabelled antibody which is specific for the epitope of interest1. A secondary antibody which carries the fluorochrome then recognizes the primary antibody and binds to it (Figure 3). Although indirect immunofluorescence is more complex and time consuming than direct immunofluorescence, it is frequently used because of its high sensitivity and it also provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In addition, a vast array of commercial secondary antibodies is available at affordable prices.
Hepatitis C virus (HCV) is a major public-health problem with 130-170 million individuals chronically infected worldwide. In order to halt the epidemic, therapy against HCV will need to be both effective and widely available. Studies focusing on safe and affordable natural product active against HCV have revealed the antiviral activity of the human Lactoferrin (hLF) protein which binds and neutralizes the circulating virion5. In the current study, investigation of hLF activity on the HCV subgenomic replicon system, which is independent from viral entry and shedding, revealed a distinct antireplicative activity of hLF against HCV. This manuscript presents a study in which immunofluorescence assays were performed to monitor the internalization of hLF, an important component of the immune system6, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus (HCV).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Wisent</td>
			<td>319-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>PAF</td>
			<td>BioShop</td>
			<td>PAR070.1</td>
			<td>Flammable solid, skin irritant, lungs and eyes.&#160;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Wisent</td>
			<td>311-425-CL</td>
			<td>Without Ca2+ &#38; Mg2+</td>
		</tr>
		<tr>
			<td>NGS</td>
			<td>Wisent</td>
			<td>053-150</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 488-labeled anti-mouse</td>
			<td>Invitrogen</td>
			<td>A11017</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 568-labeled anti-rabbit</td>
			<td>Invitrogeb</td>
			<td>A21069</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat germ agglutinin Alexa Fluor 488 conjugate (WGA)</td>
			<td>Invitrogen</td>
			<td>W11261</td>
			<td>Potentially mutagenic</td>
		</tr>
		<tr>
			<td>Anti-NS5A rabbit</td>
			<td>Abcam</td>
			<td>ab2594</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-hLF mouse</td>
			<td>Abcam</td>
			<td>ab10110</td>
			<td></td>
		</tr>
		<tr>
			<td>SlowFade</td>
			<td>Invitrogen</td>
			<td>S36937</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst stain</td>
			<td>Life Techn.</td>
			<td>H1399</td>
			<td>Potentially&#160;mutagenic&#160;and&#160;carcinogenic</td>
		</tr>
		<tr>
			<td>hLF</td>
			<td>Sigma</td>
			<td>L0520</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse visible/epifluorescence Microscope</td>
			<td>Nikon</td>
			<td>TE2000-E</td>
			<td></td>
		</tr>
		<tr>
			<td>epifluorescence/confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

immunofluorescence to monitor the cellular uptake of human lactoferrin and its associated antiviral activity against the hepatitis c virus

免疫是通常用于研究生物学的许多方面实验室技术。它通常用于可视化目标分子的细胞和组织中的分布和/或定位。免疫荧光依赖于对小区内其相应的抗原的荧光标记的抗体的特异性。直接和间接的免疫荧光方法可以用于其中依靠使用带有荧光联抗体。直接免疫是不经常使用的，因为它提供较低的信号，涉及到较高的成本和更少的灵活性。与此相反，间接免疫荧光更常用，因为其高灵敏度和提供放大的信号，因为一个以上的次级抗体可以附加到每个主抗体。在这份手稿，无论是荧光显微镜和共聚焦显微镜被用来监测人乳铁蛋白的内化，免疫的重要组成部分系统，进入肝细胞。此外，我们监测了使用免疫荧光的丙型肝炎病毒的细胞内复制的hLF的抑制潜能。都与这些方法相关的优点和缺点进行了讨论。

使用免疫荧光共聚焦miscroscope肝的Huh-7细胞系的内化外生有管理的体hLF的能力进行监测。的hLF加入到培养液中并内化被允许进行24小时，在这之后，细胞外的hLF用PBS洗涤和结合体hLF残留膜降解，5分钟胰蛋白酶处理（1ml）中。将细胞再接种，并使其重新附着的免疫荧光染色前1​​8小时。为了概括的胞质限制，将细胞的膜进行染色的488纳米缀合物的激发波长的麦胚凝集素（WGA）的荧光染料。所述的hLF?...

Watch this Scientific Journal Video about Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus at JoVE.com

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

免疫监视人乳铁蛋白的细胞摄取及其相关抗病毒活性抗丙型肝炎病毒

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

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