We would like to thank the VIB Bioimaging Core&#160;for training, support, and access to the instrument park. J.N. is supported by a PhD fellowship from the Research Foundation Flanders (FWO). Research in the J.M. group was supported by an Odysseus II Grant (G0H8618N), EOS INFLADIS (40007512), a junior research grant (G031022N) from the Research Foundation Flanders (FWO), a CRIG young investigator proof-of-concept grant, and by Ghent University. Research in the P.V. group was supported by EOS MODEL-IDI (30826052), EOS INFLADIS (40007512), FWO senior research grants (G.0C76.18N, G.0B71.18N, G.0B96.20N, G.0A9322N), Methusalem (BOF16/MET_V/007), iBOF20/IBF/039 ATLANTIS, Foundation against Cancer (F/2016/865, F/2020/1505), CRIG and GIGG consortia, and VIB.

1. Preparation of biotinylated tyramide
<ol>
	<li>Prepare biotinylated tyramide starting from biotin-tyramide. To make a 10 mM stock solution, dissolve 3.6 mg of biotin-tyramide in 1 mL of DMSO. Store the dissolved product in aliquots at &#8722;20 &#176;C to preserve the quality.</li>
</ol>
2. Maintaining HT-29 cells in culture
NOTE: ZBP1-expressing HT-29 were generated by transduction with a lentivector27 encoding human ZBP1.
<ol>
	<li>Keep ZBP1-expressing HT-29 cells in McCoy&#39;s 5A medium supplemented with L-glutamine, sodium-pyruvate, and 10% fetal bovine serum (from now on referred to as full medium) and maintain in an incubator at 37 &#176;C with 5% carbon dioxide. Ideally, use cells of a low passage number (less than 10). Leave the cells to recover for at least 5 days after the thaw cycle before the start of the experiment.</li>
	<li>To detach the cells from the culture flask, remove the medium and wash the cells with 5 mL of PBS (preheated to 37 &#176;C). Next, add the appropriate amount of trypsin/EDTA (0.05% trypsin and 0.032% EDTA, respectively, preheated to 37 &#176;C) to the cells (2 mL for a T75 flask, 3 mL for a T175 flask).</li>
	<li>Incubate the cells with trypsin/EDTA for up to 10 min in an incubator at 37 &#176;C with 5% carbon dioxide. Afterward, tap the flask and visually check if the cells detached from the flask using a microscope with 4x-20x objective magnification.</li>
	<li>If the cells are not fully detached, incubate for another 5 min at 37 &#176;C. When the cells are detached, stop the enzymatic reaction by adding 6 mL of full medium to the flask.</li>
	<li>Gather the cell suspension in a 15 mL tube. Count the cells using&#160;a trypan blue stain to assess viability; a 1:5 dilution is recommended. Only proceed with the experiment if the viability of the cells exceeds 90%.</li>
</ol>
3. Starting the experiment, seeding, and stimulation of the cells
<ol>
	<li>Seed 90,000 ZBP1-expressing HT-29 cells in full medium in a 1 cm&#178; surface area well plate that allows high end microscopy. Use an end volume of 200 &#181;L per well.</li>
	<li>Incubate the cells overnight at 37 &#176;C with 5% carbon dioxide. Take into consideration that some extra wells need to be seeded for staining controls, which is explained in more detail in step 5.6.</li>
	<li>When the cells reach 70%-80% confluency, add a necroptosis-inducing stimulus to the cells. Here, the cells were infected with HSV-1 ICP6mutRHIM (multiplicity of infection [MOI] of 5, defined as plaque forming units (pfu) divided by the number of cells; the pfu was quantified using a plaque assay on Vero cells) for 6 h, 8 h, or 10 h.</li>
	<li>As a positive control for necroptosis induction, stimulate the cells for 4 h with a necroptosis-inducing cocktail containing 30 ng/mL TNF, 20 &#181;M of the pan-caspase inhibitor zVAD-fmk, and 5 &#181;M of the SMAC mimetic BV6.</li>
	<li>As a negative control for p-RIPK3 (S227) staining, include GSK&#39;840 (1 &#181;M) to inhibit RIPK3 kinase activity and prevent the autophosphorylation of S227. Ideally, add the inhibitor in an untreated condition and after necroptosis stimulation. The inhibitor can be added simultaneously with viral infection.</li>
	<li>Prepare the stimulations in full medium, preheated to 37 &#176;C. Use an end volume of 200 &#181;L per well for all stimulations.</li>
</ol>
4. Fixing the cells
<ol>
	<li>Remove the medium and wash the cells with 200 &#181;L of 1x PBS. Then, add 150 &#181;L of 4% PFA (already equilibrated at room temperature) to the cells and incubate at room temperature for 30 min. 
	NOTE: If you are using cells that detach easily, the fixation of the cells can be optimized as follows. Remove 100 &#181;L of medium from the well, so a volume of 100 &#181;L remains on the plate. Add 100 &#181;L of 4% PFA to the cells. Incubate the cells for 5 min at room temperature. Afterward, remove the medium/fixative from the wells and replace with 150 &#181;L of 4% PFA. Incubate the cells for another 20 min at room temperature to ensure complete fixation of the cells.</li>
	<li>After fixation, remove the 4% PFA and wash the cells 3x with 200 &#181;L of 1x PBS. The samples can be stored in an excess (&#62;200 &#181;L) of 1x PBS at 4 &#176;C overnight until further processing.</li>
</ol>
5. Permeabilization and primary staining
<ol>
	<li>Remove the PBS and add 100 &#181;L of permeabilization buffer (0.5% Triton X-100 in PBS). Incubate at room temperature for 30 min.</li>
	<li>Remove the permeabilization buffer and, subsequently, wash the wells with 100 &#181;L of wash buffer (0.1% Triton X-100 in PBS). Incubate the imaging chamber with wash buffer for 5 min at room temperature on a tilting laboratory shaker (20-30 rocking motions per min).</li>
	<li>To prevent non-specific binding of primary antibodies, add 100 &#181;L of blocking medium and incubate at room temperature for 2 h. Alternatively, this blocking step may be replaced by blocking with 3% BSA, 0.1% Triton-X-100 in PBS for 1 h at room temperature.</li>
	<li>Wash the wells 3x with 100 &#181;L of wash buffer (0.1% Triton X-100 in PBS). Incubate the wash steps for 5 min at room temperature on a tilting laboratory shaker.</li>
	<li>After removing the wash buffer, add the primary antibodies to the imaging chamber and incubate overnight at 4 &#176;C. Use an end volume of 100 &#181;L to cover the well. During overnight incubation at 4&#176;C, do not place the imaging chamber on a tilting laboratory shaker. 
	NOTE: Anti p-MLKL (S358; dilution: 1:200) and anti-p-RIPK3 (S227; dilution: 1:200) share rabbit as a host species and should, therefore, not be combined in one well. To monitor the viral infection, combine a primary antibody against a viral protein of choice with p-MLKL (S358) or p-RIPK3 (S227). Here, the cells were infected with HSV-1 infection followed by ICP0 staining (dilution: 1:50, host species: mouse).</li>
	<li>Take into consideration the necessary staining controls at this point. Always include a no primary antibody condition to visualize the potential background of the TSA amplification step to set the masking threshold (see step 9). 
	&#8203;NOTE: In case of a co-staining protocol (e.g., combining p-MLKL [S358] or p-RIPK3 [S227] with an antibody against a viral protein) use single stains as well. The use of single stains is important to correct for potential bleed-through signal in other imaging channels.</li>
</ol>
6. Tyramide signal amplification (TSA)
<ol>
	<li>Remove the primary antibody mix and wash wells 3x with 100 &#181;L of wash buffer (0.1% Triton X-100 in PBS). Incubate the wash steps for 5 min at room temperature on a tilting laboratory shaker.</li>
	<li>Remove the wash buffer and add 100 &#181;L of HRP-labeled secondary antibody recognizing the species of primary antibody that needs to be amplified. To amplify the p-RIPK3 (S227) or p-MLKL (S358) signal, add 100 &#181;L of anti-rabbit-HRP and incubate for 30 min at room temperature on a tilting laboratory shaker. 
	NOTE: Only p-MLKL (S358) or p-RIPK3 (S227) will be amplified. The staining of a viral protein will be visualized using a standard indirect staining method.</li>
	<li>Afterward, wash the wells 3x with 100 &#181;L of wash buffer (0.1% Triton X-100 in PBS). Incubate the wash steps for 5 min at room temperature on a tilting laboratory shaker.</li>
	<li>In the next steps, add the biotinylated tyramide to the microscopic plate. Using the HRP-group coupled to the secondary antibodies, the enzymatic reaction will trigger the formation of tyramide radicals in close proximity to the primary target (here, p-MLKL [S358] or p-RIPK3 [S227]).</li>
	<li>To activate the enzymatic activity of HRP, add an oxidizing substrate together with the biotinylated tyramide. To achieve this, supplement the TSA buffer (0.1 M boric acid [pH 8.5]) with 0.03 M H2O2; specifically, take 5 mL of TSA buffer and add 5 &#181;L of 30% H2O2.</li>
	<li>Now, dilute the biotin-tyramide in H2O2-supplemented TSA buffer ranging from 1:1,000 to 1:20,000. 
	NOTE: The dilution factor of the biotin-tyramide needs to be optimized per batch.</li>
	<li>Remove the wash buffer from the wells and add diluted biotin-tyramide to the wells to an end volume of 100 &#181;L. Incubate at room temperature for 10 min on a tilting laboratory shaker.</li>
	<li>Next, wash the wells 3x with 100 &#181;L of wash buffer (0.1% Triton X-100 in PBS). Incubate the wash steps for 5 min at room temperature on a tilting laboratory shaker.</li>
</ol>
7. Fluorophores
NOTE: Since the signal of the primary antibody is converted to a biotin-group, p-MLKL (S358) and p-RIPK3 (S227) are visualized using streptavidin coupled to a fluorophore (fluorophore 568, dilution: 1:500). Additionally, the nuclei are stained with DAPI (5 &#181;g/mL). If a viral protein is included in the staining protocol, include a suitable fluorescently labeled secondary antibody against the host species of your primary antibody. In the representative results, a mouse anti-ICP0 was used. As a secondary antibody goat anti-mouse coupled to fluorophore 633 (dilution: 1:1,000) was included.
<ol>
	<li>Make the staining mix in wash buffer (0.1% Triton X-100 in PBS) containing the antibodies and stains mentioned above. Remove the wash buffer, add 100 &#181;L of staining mix, and incubate at room temperature for 1 h on a tilting laboratory shaker. Keep the imaging chamber shielded from light from this step onward.</li>
	<li>Next, wash the wells 2x with wash buffer (0.1% Triton X-100 in PBS). Incubate the wash steps for 5 min at room temperature on a tilting laboratory shaker.</li>
	<li>Finally, rinse the wells 2x with 1x PBS. Store the samples in an excess (&#62; 200 &#181;L) of 1x PBS until imaging. Alternatively, submerge the samples in mounting medium to preserve the staining. The imaging chamber is now ready to be visualized on a confocal microscope.</li>
</ol>
8. Imaging using a confocal microscope
<ol>
	<li>Turn on the confocal microscope and lasers at least 10 min before imaging.</li>
	<li>Use an immersion objective for sensitivity. Preferably, use 40x or 63x objective magnification. Before imaging, clean the immersion objectives with lens cleaner using appropriate cotton balls. Dirty objectives (e.g., due to dust) can result in lower quality images.</li>
	<li>Put an excess amount of oil on the bottom of the imaging chamber. Additionally, add a drop of oil on the objective of choice.</li>
	<li>Put the imaging chamber on the microscope. Find the focus field using DAPI staining or using brightfield imaging. If necessary, move around the imaging chamber to ensure proper spread of the immersion oil.</li>
	<li>Set up the necessary imaging tracks on the microscope. Depending on the microscope, the following steps may vary (Table 1). 
	NOTE: When setting up the imaging tracks, program the tracks so that the nuclear staining is measured last. The 405 nm laser may contribute to photobleaching and, thus, the loss of specific signal in all the channels.</li>
	<li>To analyze a complete cell for the presence of p-RIPK3 (S227) or p-MLKL (S358), measure z-stacks that span the height of the cell. Here, the z-stacks were made of 40 slices every 0.16 &#181;m, resulting in a range of 6.22 &#181;m.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Track</td>
			<td>Laser</td>
			<td>Beam splitter</td>
			<td>Filter</td>
		</tr>
		<tr>
			<td rowspan="2">pRIPK3 (S227) /pMLKL (S358)</td>
			<td rowspan="2">561</td>
			<td>MBS -405 +</td>
			<td rowspan="2">BP 570-620 + LP645</td>
		</tr>
		<tr>
			<td>MBS 488/561/633 + SBS SP615</td>
		</tr>
		<tr>
			<td rowspan="2">Viral Gene: ICP0</td>
			<td rowspan="2">633</td>
			<td>MBS -405 +</td>
			<td rowspan="2">BP 420-480 + LP605</td>
		</tr>
		<tr>
			<td>MBS 488/561/633 + SBS LP570</td>
		</tr>
		<tr>
			<td rowspan="2">Nucleus: DAPI</td>
			<td rowspan="2">405</td>
			<td>MBS -405 +</td>
			<td rowspan="2">BP 420-480 + BP 495-550</td>
		</tr>
		<tr>
			<td>MBS 488/561/633</td>
		</tr>
	</tbody>
</table>
Table 1: Imaging tracks for cell visualization.
9. Data analysis and quantification
<ol>
	<li>Upload the microscopic images to the software. Use the extended focus to visualize all the information of the z-stack in a 2D image.</li>
	<li>Next, program the analysis protocol in order to extract the following information: the number of cells per image, the sum of voxels showing p-RIPK3 (S227)+ or p-MLKL (S358)+ staining. A voxel represents a three-dimensional volume, defined as the three-dimensional equivalent of a pixel.</li>
	<li>To quantify the number of cells per image, segment the nucleus. To reduce noise in the segmentation results, add a size limitation to the segmentation (&#62;100 &#181;m&#179;). The number of segmented nuclei represents the number of cells in the image.</li>
	<li>To quantify the amount of p-RIPK3 (S227)+ or p-MLKL (S358)+ voxels, program a threshold-based segmentation. Set the threshold so that no signal is picked up in the no primary antibody (NP) images.</li>
	<li>Further adapt the threshold using the images from the untreated mock condition. To ensure sensitive detection of the signal increase due to necroptosis stimulation, limit the pick-up of p-RIPK3 (S227)+ or p-MLKL (S358)+ voxels in mock conditions by setting a higher threshold. Always check the programmed analysis protocol in several images from the mock condition and necroptosis-inducing viral infection.</li>
	<li>Run the analysis on all image sets. Export the voxel quantification and nucleus segmentation in a .txt file for further processing in spreadsheet software.</li>
	<li>Make a pivot table of the data showing the image name, the nucleus quantification, and the sum of detected voxels.</li>
	<li>Next, divide the sum of positive voxels by the count of cells in the image. This results in a relative value of positive voxels per cell. To visualize the fold increase, divide the relative value of p-RIPK3 (S227)+ or p-MLKL (S358)+ voxels per cell by the median of the untreated condition (mock).</li>
</ol>

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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+toolbox+for+imaging+RIPK1,+RIPK3,+and+MLKL+in+mouse+and+human+cells.">A toolbox for imaging RIPK1, RIPK3, and MLKL in mouse and human cells.</a> Cell Death and Differentiation. 28 (7), 2126-2144 (2021).</li><li>De Groote, P., 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=Generation+of+a+new+gateway-compatible+inducible+lentiviral+vector+platform+allowing+easy+derivation+of+co-transduced+cells.">Generation of a new gateway-compatible inducible lentiviral vector platform allowing easy derivation of co-transduced cells.</a> Biotechniques. 60 (5), 252-259 (2016).</li><li>Ali, M., Roback, L., Mocarski, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+1+ICP6+impedes+TNF+receptor+1-induced+necrosome+assembly+during+compartmentalization+to+detergent-resistant+membrane+vesicles.">Herpes simplex virus 1 ICP6 impedes TNF receptor 1-induced necrosome assembly during compartmentalization to detergent-resistant membrane vesicles.</a> The Journal of Biological Chemistry. 294 (3), 991-1004 (2019).</li><li>Mandal, P., 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=RIP3+induces+apoptosis+independent+of+pronecrotic+kinase+activity.">RIP3 induces apoptosis independent of pronecrotic kinase activity.</a> Moleular Cell. 56 (4), 481-495 (2014).</li><li>Zhang, T., 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=Influenza+virus+Z-RNAs+induce+ZBP1-mediated+necroptosis.">Influenza virus Z-RNAs induce ZBP1-mediated necroptosis.</a> Cell. 180 (6), 1115-1129 (2020).</li><li>Garnish, S. E., 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=Conformational+interconversion+of+MLKL+and+disengagement+from+RIPK3+precede+cell+death+by+necroptosis.">Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis.</a> Nature Communications. 12 (1), 2211 (2021).</li><li>Clay, H., Ramakrishnan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+fluorescent+in+situ+hybridization+in+zebrafish+embryos+using+tyramide+signal+amplification.">Multiplex fluorescent in situ hybridization in zebrafish embryos using tyramide signal amplification.</a> Zebrafish. 2 (2), 105-111 (2005).</li><li>Parra, E. 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=Procedural+requirements+and+recommendations+for+multiplex+immunofluorescence+tyramide+signal+amplification+assays+to+support+translational+oncology+studies.">Procedural requirements and recommendations for multiplex immunofluorescence tyramide signal amplification assays to support translational oncology studies.</a> Cancers. 12 (2), 255 (2020).</li></ol>

The authors have nothing to disclose.

This immunofluorescent staining protocol describes the use of tyramide signal amplification (TSA) to increase the sensitivity for signaling events of the human necroptotic signaling pathway that are difficult to detect, including the phosphorylation of RIPK3 and MLKL26. The inclusion of a TSA step significantly improves the detection threshold of p-RIPK3 (S227) and p-MLKL (S358) and increases the sensitivity of p-MLKL (S358) straining. TSA revealed a p-RIPK3 (S227) signal already present in the mo...

Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) are central regulators of necroptotic cell death1,2. Necroptosis is a lytic and inflammatory form of regulated cell death involved in antiviral immunity and autoinflammation. Necroptosis of virus-infected cells immediately shuts down virus replication. Cell lysis following necroptosis induction also releases damage-associated molecular patterns, which stimulate antiviral immunity3,4. Necroptosis is initiated by the activation of RIPK3 following RIP homotypic interaction motif (RHIM)-mediated interactions with one of three upstream activating molecules: RIPK1 (upon TNF receptor 1 [TNFR1] engagement), TIR-domain-containing adapter-inducing interferon-&#946; (TRIF; upon Toll-like receptor 3 and 4 engagement), or the antiviral nucleic acid sensor Z-DNA binding protein 1 (ZBP1)1,2. Necroptosis signaling proceeds through a series of phosphorylation events beginning with the autophosphorylation of RIPK3. The autophosphorylation of human RIPK3 at serine (S)227 inside its kinase domain is a prerequisite for necroptosis by enabling the interaction with MLKL and is commonly used as a biochemical marker for human RIPK3 activation and necroptotic cell death1,5. Once activated, RIPK3 phosphorylates the activation loop of MLKL at threonine (T)357 and S3581. This causes a change in MLKL conformation, resulting in exposure of the N-terminal four helix bundle domain. MLKL then oligomerizes and traffics to the cell membrane where it forms a pore through the insertion of the exposed four helix bundles in the lipid bilayer, eventually leading to cell death2,6.
ZBP1 is an antiviral nucleic acid sensor that recognizes left-handed Z-form nucleic acids including double-stranded RNA in the Z-conformation (Z-RNA). Z-RNA binding occurs via two Z&#945;-domains positioned at the N-terminus of ZBP1. Z-RNA accumulating during RNA and DNA virus infection is thought to directly engage ZBP17,8. Activated ZBP1 recruits RIPK3 through its central RHIMs and induces regulated cell death, including necroptosis9,10. Viruses have adopted numerous escape mechanisms to counteract ZBP1-induced host cell necroptosis11. For example, the herpes simplex virus 1 (HSV-1) ribonucleotide reductase subunit 1, known as ICP6 and encoded by UL39, harbors RHIM at its N-terminus that interferes with ZBP1-mediated RIPK3 activation in human cells12,13,14,15. ZBP1 not only restricts viral replication, but mouse studies have shown that ZBP1 activation causes inflammatory diseases and stimulates cancer immunity16,17,18,19,20,21. Protocols that detect signaling events occurring during ZBP1-induced necroptosis in human cells are, therefore, valuable to assess the role of ZBP1 in these processes.
Tyramide signal amplification (TSA), also referred to as catalyzed reporter deposition (CARD), has been developed to improve the limit of detection and signal-to-noise ratio in antibody-based immunoassays. During TSA, any primary antibody can be used to detect the antigen of interest. Horseradish peroxidase (HRP), coupled to a secondary antibody, catalyzes the local build-up of biotinylated tyramide radicals in the presence of hydrogen peroxide. These activated biotin-tyramide radicals then react with proximal tyrosine residues to form covalent bonds. Potential tyramide-biotin substrates include the antigen itself, the primary and secondary antibodies, and neighboring proteins. Thus, while TSA significantly improves the sensitivity of the assay, some of its spatial resolution is lost. In a final step, biotin molecules are detected using fluorescently labeled streptavidin. The HRP reaction deposits many tyramide-biotin molecules on or near the antigen of interest. This greatly increases the number of streptavidin-fluorochrome binding sites, thereby greatly amplifying the sensitivity of the assay (Figure 1). Alternatively, tyramide can be directly coupled to a fluorochrome, eliminating the need for streptavidin-coupled fluorophores. Protein immunohistochemistry and DNA/RNA in situ hybridization were among the first methods whereby TSA was employed to improve signal intensities22,23. More recently, TSA has been combined with intracellular flow cytometry24 and mass spectrometry25.
Here, we present a protocol to detect serine 227 phosphorylated human RIPK3 (p-RIPK3 [S227]) and phosphorylated human MLKL (p-MLKL [S358]) upon the activation of ZBP1 by HSV-1 infection using immunofluorescence microscopy. We use a necroptosis-sensitive HT-29 human colorectal adenocarcinoma cell line that was transduced to stably express human ZBP1. These cells were infected with an HSV-1 strain expressing a mutant ICP6 protein (HSV-1 ICP6mutRHIM) in which four core amino acids within the viral RHIM (VQCG) were replaced by alanines (AAAA), thereby rendering the ICP6 unable to block ZBP1-mediated necroptosis13,14,15. To overcome the problem of the low signal-to-noise ratio of the currently commercially available antibodies directed against p-RIPK3 and p-MLKL in immunostaining26, we perform a tyramide signal amplification (TSA) step (Figure 1), which results in the robust detection of human p-RIPK3 (S227) and improves the detection sensitivity of human p-MLKL (S358) by an order of magnitude.

<table><tbody><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-rabbit HRP</td><td>Agilent Technologies Belgium</td><td>K4002</td><td>Envision+ System-HRP Labelled Polymer anti-rabbit</td></tr><tr><td>Goat anti-mouse DyLight 633</td><td>Thermofisher</td><td>35513</td><td>Secundary antibody</td></tr><tr><td>HSV-1 ICP0</td><td>Santa Cruz</td><td>sc-53070</td><td>Mouse anti-ICP0(HSV-1) antibody</td></tr><tr><td>IAV-PR8 mouse serum</td><td>In house production</td><td>xx</td><td>Mouse anti-IAV-PR8 polyclonal antibody</td></tr><tr><td>pMLKL</td><td>Abcam</td><td>ab187091</td><td>Rabbit anti-MLKL-phospho S358 antibody</td></tr><tr><td>pRIPK3</td><td>Abcam</td><td>ab209384</td><td>Rabbit anti-RIPK3-phospho S227 antibody</td></tr><tr><td>&lt;strong&gt;Fluorophores&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DAPI</td><td>Thermofisher</td><td>D21490</td><td>To visualise the nucleus of the cells</td></tr><tr><td>Streptavidin coupled to Alexa Fluor 568</td><td>Thermofisher</td><td>S11226</td><td>To visulalise biotin molecules</td></tr><tr><td>&lt;strong&gt;Compounds&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>30% H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>H1009</td><td>Oxidising substrate, necessary for HRP activity</td></tr><tr><td>4% PFA</td><td>SANBIO</td><td>AR1068</td><td>To fix/crosslink the cells</td></tr><tr><td>Biotinyl-tyramide</td><td>R&amp;amp;D Systems</td><td>6241</td><td>To amplify signal, HRP substrate</td></tr><tr><td>BV-6</td><td>Selleckchem</td><td>S7597</td><td>BV6 IAP Inhibitor</td></tr><tr><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>For cell culture: to detach the cells</td></tr><tr><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>8.0 g/L NaCl</td></tr><tr><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>0.4 g/L disodium salt of EDTA</td></tr><tr><td>EDTA 0.04%</td><td>In house formulation</td><td></td><td>1.1 g/L Na2HPO4</td></tr><tr><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>0.2 g/L NaH2PO4</td></tr><tr><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>0.2 g/L KCl</td></tr><tr><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>0.2 g/L Glucose</td></tr><tr><td>Fetal Bovine serum</td><td>TICO</td><td>FBS EU XXX</td><td>For cell culture, maintaining cell culture; lot number: 90439</td></tr><tr><td>GSK&#039;840</td><td>Aobious</td><td>AOB0917</td><td>RIPK3 kinase inhibitor</td></tr><tr><td>L-Glutamine</td><td>Sigma-Aldrich</td><td>G7513</td><td>For cell culture, maintaining cell culture</td></tr><tr><td>MAXblock</td><td>Active Motif</td><td>15252</td><td>Blocking solution</td></tr><tr><td>PBS</td><td>Gibco</td><td>10444402</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>S8636</td><td>For cell culture, maintaining cell culture</td></tr><tr><td>TNF-&amp;alpha;</td><td>In house production</td><td>-</td><td>Signaling molecule, able to trigger cell death in combination with BV6 and zVAD</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T8787-50ML</td><td>To permeabilise the cells</td></tr><tr><td>Trypan blue</td><td>Merck</td><td>11732</td><td>For cell counting, used as live/dead marker at 0,1%</td></tr><tr><td>Trypsine</td><td>Sigma-Aldrich</td><td>T4424</td><td>For cell culture: to detach the cells</td></tr><tr><td>zVAD</td><td>Bachem</td><td>BACE4026865.0005</td><td>Z-Val-Ala-DL-Asp-fluoromethylketone</td></tr><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;micro;-Slide 8 well high glass bottom</td><td>iBidi</td><td>80807</td><td>To culture the cells</td></tr><tr><td>Cotton Preping Balls-size medium</td><td>Electron Microscopy Sciences</td><td>71001-10</td><td>To clean the objectives</td></tr><tr><td>Immersol 518 F / 30 &amp;deg;C</td><td>ZEISS</td><td>444970-9000-000</td><td>To visualise the sample at high magnifications</td></tr><tr><td>Lens Cleaner</td><td>ZEISS</td><td>000000-0105-200</td><td>To clean the objectives</td></tr><tr><td>LSM880 Fast Airyscan confocal microscope</td><td></td><td></td><td>To visualise the sample</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Excel</td><td>Office</td><td>xx</td><td>To process the data</td></tr><tr><td>Prism 9</td><td>Graphpad</td><td>xx</td><td>To analyse the data- statistical testing and graph generation</td></tr><tr><td>Volocity 6.3</td><td>Volocity</td><td>xx</td><td>To perform quantifications</td></tr><tr><td>Zen black</td><td>ZEISS</td><td>xx</td><td>To aquire and process images</td></tr><tr><td>Zen blue</td><td>ZEISS</td><td>xx</td><td>To visualise images</td></tr><tr><td>&lt;strong&gt;Viruses&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HSV-1 (mutRHIM) F strain</td><td>produced by&amp;nbsp; Dr. Jiahuai Han</td><td>in house replication</td><td>HSV-1 as a trigger for necroptosis; RHIM core domain of UL39/ICP6 is mutated (VQCG&gt;AAAA)</td></tr><tr><td>HSV-1 (WT) F strain</td><td>Produced by Dr. Jiahuai Han</td><td>in house replication</td><td>HSV-1 (WT) as a negative control for necroptosis induction (ICP6 inhibition)</td></tr><tr><td>IAV PR8</td><td>in house stock</td><td>in house replication</td><td>IAV as a trigger for necroptosis</td></tr></tbody></table>

tyramide signal amplification for the immunofluorescent staining of zbp1-dependent phosphorylation of ripk3 and mlkl after hsv-1 infection in human cells

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.

The immunofluorescent detection of MLKL phosphorylation and especially RIPK3 phosphorylation in human cells is technically challenging26. We here present an improved staining protocol for human p-RIPK3 (S227) and p-MLKL (S358) upon the activation of ZBP1. The protocol includes a TSA step to improve the detection limit and sensitivity of the fluorescent signals. To validate the method, a side-by-side comparison of the TSA-mediated immunofluorescence with standard indirect fluorescent staining of bo...

Watch this Scientific Journal Video about Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells at J

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

Tyramide signal amplification during immunofluorescent staining enables the sensitive detection of phosphorylated RIPK3 and MLKL during ZBP1-induced necroptosis after HSV-1 infection.

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical ...

tyramide-signal-amplification-for-immunofluorescent-staining-zbp1

Research

JoVE Journal

Immunology and Infection

2.1K Views.  VIB-UGent Center for Inflammation Research. Robuuste visualisatie van gefosforyleerde RIPK3 en MLKL, twee belangrijke signaleringsgebeurtenissen tijdens necroptose-inductie, is technisch uitdagend geweest. Het gepresenteerde tyramide-amplificatieprotocol maakt gevoelige detectie van deze twee moleculen mogelijk. De signaalversterkingsstap verlaagt de detectiedrempel, waardoor robuuste en reproduceerbare detectie van gefosforyleerde RIPK3 en MLKL mogelijk is.Begin met het zaaien van de 90.000 ZBP1-expresserende HT-29-cellen in he...

Video: Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Neuroscience

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience ...

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 understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

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 ...

Biology

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.

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 ...

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

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