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. Cellen Voorbereiding en behandeling <ol><li> In een 24-wells plaat, zet een glazen deksel aan de onderzijde van de putjes. Was elke well met fosfaat gebufferde zoutoplossing (PBS). </li><li> Seed Huh-7 cellen ondersteunen subgenomische HCV replicon in elk putje tot een dichtheid van 5 x 10 4 cellen / putje. Als er geen behandeling te doen, kunnen de cellen worden gezaaid met een hogere dichtheid. Het kweekmedium Dulbecco&#39;s Modified Eagle Medium (DMEM) aangevuld met 10% foetaal runderserum (FBS), 2 mM...

<ol>
	<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>

De HCV-epidemie blijft een wereldwijde bedreiging, met 80% van de nieuw geïnfecteerde patiënten ontwikkelen een chronische infectie, waardoor ze het risico van cirrose, leverfalen of leverkanker. Direct werkende antivirale middelen gericht replicatie en rijping HCV vertegenwoordigen prime anti-HCV middelen, zoals onlangs aangetoond door de goedkeuring van twee NS3 N-terminus proteaseremmers (Boceprevir en Telaprevir). Het hLF anti-HCV-activiteit wordt momenteel verondersteld zich te gedragen als een virale binnenkomst...

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

Immunofluorescentie is een laboratorium techniek die vaak gebruikt om vele aspecten van de biologie te bestuderen. Het wordt meestal gebruikt om de verdeling en / of lokalisatie van een doelwitmolecuul in cellen en weefsels zichtbaar. Immunofluorescentie gebaseerd op de specificiteit van fluorescent-gelabelde antilichamen tegen het overeenkomstige antigeen in een cel. Zowel directe als indirecte immunofluorescentie benaderingen kunnen worden toegepast die gebaseerd zijn op het gebruik van antilichamen gekoppeld aan een fluorochroom. Directe immunofluorescentie wordt minder vaak gebruikt omdat het lagere signaal impliceert hogere kosten en minder flexibiliteit. Daarentegen wordt indirecte immunofluorescentie vaker gebruikt vanwege de hoge gevoeligheid en levert een versterkt signaal aangezien meerdere secundair antilichaam kan hechten aan elke primaire antilichaam. In dit manuscript werden beide epifluorescentie microscopie en confocale microscopie gebruikt om de internalisering van menselijk lactoferrine, een belangrijke component van de immune bewakensysteem, in de levercellen. Bovendien hebben we bewaakt de remmende potentie van hLF de intracellulaire replicatie van het hepatitis C virus middels immunofluorescentie. Zowel de voor- en nadelen verbonden aan deze benaderingen besproken.

Het vermogen van de lever Huh-7 cellijn te internaliseren exogeen toegediende hLF werd gevolgd met behulp van immunofluorescentie op een confocale miscroscope. hLF werd aan het kweekmedium toegevoegd en de internalisatie liet men gedurende 24 uur, waarna werd extracellulair hLF gewassen met PBS en overgebleven membraangebonden hLF werd afgebroken met een 5 min trypsine behandeling (1 ml). De cellen werden opnieuw uitgezet en liet opnieuw hechten 18 uur voor immunofluorescentiekleuring. Om het cytoplasmatische grenzen aa...

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.

Immunofluorescentie te zien op de cellulaire opname van humaan lactoferrine en de Associated antivirale activiteit tegen het hepatitis C virus

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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