Name: single-cell multiplexed fluorescence imaging to visualize viral nucleic acids and proteins and monitor hiv, htlv, hbv, hcv, zika virus, and influenza infection
Uploaded: 2020-10-29T14:00:00
Description: Watch this Scientific Journal Video about Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infectio

This work was supported in whole or in part by the National Institutes of Health (R01 AI121315, R01 AI146017, R01 AI148382, R01 AI120860, R37 AI076119, and U54 AI150472). We thank Dr. Raymond F. Schinazi and Sadie Amichai for providing cells infected with influenza A Virus.

1. Seed cells (Suspension vs. Adherent Cells) on coverslips or chamber slides (protocol presented uses coverslips).
<ol>
	<li>Seeding of suspension cells
	<ol>
		<li>Prepare poly-D-lysine (PDL) coated coverslips (facilitates adherence of suspension cells to coverslip) by first incubating coverslips in ethanol (EtOH) for 5 minutes (sterilizes coverslips and removes any residue). Then wash 2x in phosphate buffered saline (PBS) before incubating coverslips in PDL (20 &#181;g/mL) for 30 minutes (min) at room tempe...

<ol>
	<li>Zheng, Q., Lavis, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+photostable+fluorophores+for+molecular+imaging.">Development of photostable fluorophores for molecular imaging.</a> Current Opinion in Chemical Biology. 39, 32-38 (2017).</li><li>Marini, B., 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=Nuclear+architecture+dictates+HIV-1+integration+site+selection.">Nuclear architecture dictates HIV-1 integration site selection.</a> Nature. 521 (7551), 227-231 (2015).</li><li>Puray-Chavez, M., 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=Multiplex+single-cell+visualization+of+nucleic+acids+and+protein+during+HIV+infection.">Multiplex single-cell visualization of nucleic acids and protein during HIV infection.</a> Nature Communications. 8 (1), 1882 (2017).</li><li>Ukah, O. B., 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=Visualization+of+HIV-1+RNA+Transcription+from+Integrated+HIV-1+DNA+in+Reactivated+Latently+Infected+Cells.">Visualization of HIV-1 RNA Transcription from Integrated HIV-1 DNA in Reactivated Latently Infected Cells.</a> Viruses. 10 (10), (2018).</li><li>Liu, D., 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=Visualization+of+Positive+and+Negative+Sense+Viral+RNA+for+Probing+the+Mechanism+of+Direct-Acting+Antivirals+against+Hepatitis+C+Virus.">Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus.</a> Viruses. 11 (11), (2019).</li><li>Achuthan, V., 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=Capsid-CPSF6+Interaction+Licenses+Nuclear+HIV-1+Trafficking+to+Sites+of+Viral+DNA+Integration.">Capsid-CPSF6 Interaction Licenses Nuclear HIV-1 Trafficking to Sites of Viral DNA Integration.</a> Cell Host &amp; Microbe. 24 (3), 392-404 (2018).</li><li>Wang, F., 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=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> Journal of Molecular Diagnostics. 14 (1), 22-29 (2012).</li><li>Xia, C., Babcock, H. P., Moffitt, J. R., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+detection+of+RNA+using+MERFISH+and+branched+DNA+amplification.">Multiplexed detection of RNA using MERFISH and branched DNA amplification.</a> Scientific Reports. 9 (1), 7721 (2019).</li><li>Player, A. N., Shen, L. P., Kenny, D., Antao, V. P., Kolberg, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-copy+gene+detection+using+branched+DNA+(bDNA)+in+situ+hybridization.">Single-copy gene detection using branched DNA (bDNA) in situ hybridization.</a> Journal of Histochemistry and Cytochemistry. 49 (5), 603-612 (2001).</li><li>Battich, N., Stoeger, T., Pelkmans, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+transcriptomics+in+thousands+of+single+human+cells+at+single-molecule+resolution.">Image-based transcriptomics in thousands of single human cells at single-molecule resolution.</a> Nature Methods. 10 (11), 1127-1133 (2013).</li><li>Bushnell, 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=ProbeDesigner:+for+the+design+of+probesets+for+branched+DNA+(bDNA)+signal+amplification+assays.">ProbeDesigner: for the design of probesets for branched DNA (bDNA) signal amplification assays.</a> Bioinformatics. 15 (5), 348-355 (1999).</li><li>Stultz, R. D., Cenker, J. J., McDonald, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+HIV-1+Genomic+DNA+from+Entry+through+Productive+Infection.">Imaging HIV-1 Genomic DNA from Entry through Productive Infection.</a> Journal of Virology. 91 (9), (2017).</li><li>Brown, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+nuclear+proteins+together+with+transcribed+and+inactive+genes+in+structurally+preserved+cells.">Visualizing nuclear proteins together with transcribed and inactive genes in structurally preserved cells.</a> Methods. 26 (1), 10-18 (2002).</li><li>Francis, A. C., 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=HIV-1+replication+complexes+accumulate+in+nuclear+speckles+and+integrate+into+speckle-associated+genomic+domains.">HIV-1 replication complexes accumulate in nuclear speckles and integrate into speckle-associated genomic domains.</a> Nature Communications. 11 (1), 3505 (2020).</li><li>Dharan, A., Bachmann, N., Talley, S., Zwikelmaier, V., Campbell, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+pore+blockade+reveals+that+HIV-1+completes+reverse+transcription+and+uncoating+in+the+nucleus.">Nuclear pore blockade reveals that HIV-1 completes reverse transcription and uncoating in the nucleus.</a> Nature Microbiology. 5 (9), 1088-1095 (2020).</li></ol>

Simultaneous visualization of RNA, DNA, and protein often requires extensive optimization. Two commonly used methods are 5-ethynyl-2-deoxyuridine (EdU) labeling and DNA FISH. EdU labeling has been applied to visualize viral DNA and protein simultaneously, as EdU is incorporated in nascent DNA and subsequently labeled with azide-containing fluorescent dyes via click chemistry. EdU labeling can thus be used to monitor native virus replication kinetics of DNA viruses or viruses with DNA templates for replication<su...

While thousands of commercial antibodies are available to specifically label proteins via conventional immunostaining approaches, and while fusion proteins can be engineered with photo-optimized fluorescent tags for tracking multiple proteins in a sample1, microscopic visualization of protein is often not compatible with conventional DNA fluorescence in situ hybridization (FISH)2. Technical limitations in simultaneous visualization of DNA, RNA, and protein using fluorescence-based approaches have hindered in-depth understanding of virus replication. Tracking both viral nucleic acid and protein during the course of infection allows virologists to visualize fundamental processes that underly virus replication and assembly3,4,5,6.
We have developed an imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and Protein (MICDDRP)3, which utilizes branched DNA (bDNA) in situ technology to improve the sensitivity of nucleic acid detection7,8,9. In addition, this method utilizes paired probes for enhanced specificity. bDNA sequence-specific probes use branching preamplifier and amplifier DNAs to produce an intense and localized signal, improving upon previous hybridization methods that relied on targeting repeated regions in the DNA9. Infected cells in a clinical context often do not contain abundant viral genetic material, providing a commodity for a sensitive method for fluorescent nucleic acid detection in diagnostic settings. The commercialization of bDNA technology through approaches such as RNAscope7 and ViewRNA10 have filled this niche. The sensitivity of bDNA fluorescence imaging also has important utility in cell biology, allowing detection of scarce nucleic acid species in cell culture models. The vast improvement of sensitivity makes bDNA-based imaging methods suitable for studying viruses. A potential shortcoming, however, is that these methods focus on visualizing RNA or RNA and protein. All replicating cells and many viruses have DNA genomes or form DNA during their replication cycle, making methods capable of imaging both RNA and DNA, as well as protein, highly desirable.
In the MICDDRP protocol, we perform bDNA FISH for detection of viral nucleic acid using the RNAscope method, with modifications7. One of the major modifications to this protocol is optimization of protease treatment following chemical fixation. Protease treatment facilitates removal of proteins bound to nucleic acid to improve probe hybridization efficiency. Protease treatment is followed by incubation with branched oligonucleotide probe(s). After application of bDNA probe(s), samples are washed and subsequently incubated with signal pre-amplifier and amplifier DNAs. Multiplexed in situ hybridization (ISH), labeling of multiple gene targets, requires target probes with different color channels for spectral differentiation7. Incubation with DNA amplifiers is followed by immunofluorescence (IF).
bDNA ISH imparts improvements in signal-to-noise by amplification of target-specific signals, with a reduction to background noise from non-specific hybridization events7,11. Target probes are designed using software programs publicly available that predict the probability of non-specific hybridization events, as well as calculate melting temperature (Tm) of the probe-target hybrid7,11. Target probes contain an 18- to 25-base region complementary to the target DNA/RNA sequence, a spacer sequence, and a 14-base tail sequence. A pair of target probes, each with a distinct tail sequence, hybridize to a target region (spanning ~50 bases). The two tail sequences form a hybridization site for the pre-amplifier probes, which contain 20 binding sites for the amplifier probes, which, in addition contain 20 binding sites for the label probe. As an example, a one kilobase (kb) region on the nucleic acid molecule is targeted by 20 probe pairs, creating a molecular scaffold for sequential hybridization with the preamplifier, amplifier, and label probe. This can thus lead to a theoretical yield of 8000 fluorescent labels per nucleic acid molecule, enabling detection of single molecules and vast improvements over conventional FISH approaches7 (See Figure 1A for schematic of bDNA signal amplification). To set up probes for multiplexed ISH, each target probe must be in a different color channel (C1, C2, or C3). These target probes with different color channels possess distinct 14-base tail sequences. These tail sequences will bind distinct signal amplifiers with different fluorescent probes, thus enabling facile spectral differentiation across multiple targets. In the presented Protocol, Table 4 in Step 9, provides further information on fluorescently labeling target probes. In addition, Figure 2 and Figure 3 provide examples of how we chose the appropriate Amplifier 4-FL (A, B, or C) (fluorescent probe &#38; the final hybridization step) to achieve specific fluorescent labeling of multiple viral nucleic acid targets following HIV-1 and HTLV-1 infections.
We have demonstrated several applications of simultaneous fluorescence visualization of RNA, DNA, and proteins, observing critical stages of virus replication with high spatiotemporal resolution3,4,5. For example, simultaneous single-cell visualization of viral RNA, cytoplasmic and nuclear DNA, and protein have allowed us to visualize key events during HIV-1 infection, including following RNA containing cores in the cytoplasm prior to nuclear entry and integration of proviral DNA3. In addition, we have applied MICDDRP to characterize the effects of host factors and drug treatment on viral infection and replication4,5. In Ukah et al., we tracked reactivation of HIV-1 transcription in latency cell models treated with different latency-reversing agents to visualize HIV transcription and latency reversal4. In addition, MICDDRP can allow us to visualize phenotypic changes associated with antiviral inhibition attributed to small molecule treatment or host factor restriction. As a proof of concept to the robustness and broad applicability of our approach, we have demonstrated that we can use modifications of our protocol to efficiently label viral nucleic acid to follow infection not only in human immunodeficiency virus (HIV)-1, but also human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZIKV), and influenza A virus (IAV). As the HIV-1 life cycle consists of both viral DNA and RNA species, we have performed the majority of our optimization of MICDDRP following HIV-1 replication kinetics. However, in addition, we have demonstrated that we can track synthesis of different viral RNA transcripts of either or both sense (+) and antisense (-) strandedness in viruses such as ZIKV, IAV, HBV, and HCV to monitor viral transcription and replication3,4,5,6. The studies aim to improve the mechanistic understanding of several viral processes and serve as a guideline to implement this fluorescence imaging technology to a broad range of cellular models.

<table>
	<tbody>
		<tr>
			<td>4% PFA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50% dextran sulfate</td>
			<td>Amreso</td>
			<td>198</td>
			<td>For DNA hybridization buffer</td>
		</tr>
		<tr>
			<td>50X wash buffer</td>
			<td>ACD Bio</td>
			<td>320058</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier 1-FL</td>
			<td>ACD Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier 2-FL</td>
			<td>ACD Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier 3-FL</td>
			<td>ACD Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier 4-FL</td>
			<td>ACD Bio</td>
			<td></td>
			<td>Consult Amp-4 table in protocol</td>
		</tr>
		<tr>
			<td>Anti-HCV NS5a antibody</td>
			<td>Abcam</td>
			<td>ab13833</td>
			<td>Mouse monoclonal; works with HCV genotypes 1a, 1b, 3, and 4</td>
		</tr>
		<tr>
			<td>Anti-HIV-1 p24 monoclonal antibody</td>
			<td>NIH AIDS Reagent Program</td>
			<td>3537</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mov10 antibody</td>
			<td>Abcam</td>
			<td>ab80613</td>
			<td>Rabbit polyclonal</td>
		</tr>
		<tr>
			<td>Anti-PB1 antibody</td>
			<td>GeneTex</td>
			<td>GTX125923</td>
			<td>Antibody against flu protein</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td></td>
			<td></td>
			<td>Blocking reagent for immunostaining</td>
		</tr>
		<tr>
			<td>Cell media with supplements</td>
			<td></td>
			<td></td>
			<td>Media appropriate for cell model</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ACD Bio</td>
			<td></td>
			<td>Nuclear stain (RNAscope kit from ACD Bio)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (1X PBS)</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td>No calcium and magnesium</td>
		</tr>
		<tr>
			<td>Ethylene carbonate</td>
			<td>Sigma</td>
			<td>E26258</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td></td>
			<td></td>
			<td>Use specific FBS based on what serum secondary antibody was raised in (e.g goat FBS)</td>
		</tr>
		<tr>
			<td>Fisherbrand colorfrost plus microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-17/18/19</td>
			<td>Precleaned</td>
		</tr>
		<tr>
			<td>HCV-GT2a-sense-C2 probe</td>
			<td>ACD Bio</td>
			<td>441371</td>
			<td>HCV(+) sense RNA probe</td>
		</tr>
		<tr>
			<td>HIV-gagpol-C1</td>
			<td>ACD Bio</td>
			<td>317701</td>
			<td>HIV-1 cDNA probe</td>
		</tr>
		<tr>
			<td>HIV-nongagpol-C3</td>
			<td>ACD Bio</td>
			<td>317711-C</td>
			<td>HIV-1 RNA probe</td>
		</tr>
		<tr>
			<td>HybEZ hybridization oven</td>
			<td>ACD Bio</td>
			<td>321710/321720</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmEdge hydrophobic barrier pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td></td>
			<td></td>
			<td>For immobolizing coverslip to slide prior to protease treatment</td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>Ambion</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-d-lysine (PDL)</td>
			<td></td>
			<td></td>
			<td>Coat coverslips in 20 &#181;g/mL of PDL for 30 minutes</td>
		</tr>
		<tr>
			<td>Probe diluent</td>
			<td>ACD Bio</td>
			<td>300041</td>
			<td>For diluting RNA C2 or C3 probes</td>
		</tr>
		<tr>
			<td>Prolong gold antifade</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease III</td>
			<td>ACD Bio</td>
			<td>322337</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope&#174;&#160;Probe- V-Influenza-H1N1-H5N1-NP</td>
			<td>ACD Bio</td>
			<td>436221</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Qiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td></td>
			<td></td>
			<td>For DNA hybridization buffer</td>
		</tr>
		<tr>
			<td>Sodium citrate, pH 6.2</td>
			<td></td>
			<td></td>
			<td>For DNA hybridization buffer</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td></td>
			<td></td>
			<td>For DNA hybridization buffer and PBS-T</td>
		</tr>
		<tr>
			<td>V-HBV-GTD</td>
			<td>ACD Bio</td>
			<td>441351</td>
			<td>Total HBV RNA</td>
		</tr>
		<tr>
			<td>V-HBV-GTD-01-C2</td>
			<td>ACD Bio</td>
			<td>465531-C2</td>
			<td>HBV pgRNA probe</td>
		</tr>
		<tr>
			<td>V-HCV-GT2a probe</td>
			<td>ACD Bio</td>
			<td>441361</td>
			<td>HCV(-) sense RNA probe</td>
		</tr>
		<tr>
			<td>V-HTLV-HBZ-sense-C3</td>
			<td>ACD Bio</td>
			<td>495071-C3</td>
			<td>HTLV-1 (-) sense RNA probe targetting HBZ</td>
		</tr>
		<tr>
			<td>V-HTLV1-GAG-C2</td>
			<td>ACD Bio</td>
			<td>495051-C2</td>
			<td>HTLV-1 DNA probe</td>
		</tr>
		<tr>
			<td>V-HTLV1-GAG-POL-sense</td>
			<td>ACD Bio</td>
			<td>495061</td>
			<td>HTLV-1 (+) sense RNA probe</td>
		</tr>
		<tr>
			<td>V-Influenza-H1N1-H5N1-NP</td>
			<td>ACD Bio</td>
			<td>436221</td>
			<td>IAV RNA probe</td>
		</tr>
		<tr>
			<td>V-ZIKA-pp-O2</td>
			<td>ACD Bio</td>
			<td>464531</td>
			<td>Zika(+) sense RNA probe</td>
		</tr>
		<tr>
			<td>V-ZIKA-pp-O2-sense-C2</td>
			<td>ACD Bio</td>
			<td>478731-C2</td>
			<td>Zika(-) sense RNA probe</td>
		</tr>
	</tbody>
</table>

single-cell multiplexed fluorescence imaging to visualize viral nucleic acids and proteins and monitor hiv, htlv, hbv, hcv, zika virus, and influenza infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

A schematic of MICDDRP is depicted in Figure 1. Labeling of DNA and RNA is followed by immunostaining. The use of branching amplifiers increases signal, allowing detection of single nucleic acid molecules.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61843/61843fig1v5.jpg" /> 
Figure 1: Schematic of MICDDRP and step-by-step workflow.&#160;(A) bDNA signal is amplified via branch...

Watch this Scientific Journal Video about Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infectio

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

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Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as ...