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 encodi...

<ol>
	<li>Meng, Y., Sandow, J. J., Czabotar, P. E., Murphy, J. 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+regulation+of+necroptosis+by+post-translational+modifications.">The regulation of necroptosis by post-translational modifications.</a> Cell Death &amp; Differentiation. 28 (3), 861-883 (2021).</li><li>Petrie, E. J., Czabotar, P. E., Murphy, J. 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+structural+basis+of+necroptotic+cell+death+signaling.">The structural basis of necroptotic cell death signaling.</a> Trends in Biochemical Sciences. 44 (1), 53-63 (2019).</li><li>Mocarski, E. S., Guo, H., Kaiser, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necroptosis:+The+Trojan+horse+in+cell+autonomous+antiviral+host+defense.">Necroptosis: The Trojan horse in cell autonomous antiviral host defense.</a> Virology. 479-480, 160-166 (2015).</li><li>Nailwal, H., Chan, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necroptosis+in+anti-viral+inflammation.">Necroptosis in anti-viral inflammation.</a> Cell Death &amp; Differentiation. 26 (1), 4-13 (2019).</li><li>Meng, Y., 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=Human+RIPK3+maintains+MLKL+in+an+inactive+conformation+prior+to+cell+death+by+necroptosis.">Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis.</a> Nature Communications. 12 (1), 6783 (2021).</li><li>Samson, A. L., Garnish, S. E., Hildebrand, J. M., Murphy, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location,+location,+location:+A+compartmentalized+view+of+TNF-induced+necroptotic+signaling.">Location, location, location: A compartmentalized view of TNF-induced necroptotic signaling.</a> Science Signalling. 14 (668), (2021).</li><li>Koehler, H., 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=Vaccinia+virus+E3+prevents+sensing+of+Z-RNA+to+block+ZBP1-dependent+necroptosis.">Vaccinia virus E3 prevents sensing of Z-RNA to block ZBP1-dependent necroptosis.</a> Cell Host Microbe. 29 (8), 1266-1276 (2021).</li><li>Balachandran, S., 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=Viral+Z-RNA+triggers+ZBP1-dependent+cell+death.">Viral Z-RNA triggers ZBP1-dependent cell death.</a> Current Opinion in Virology. 51, 134-140 (2021).</li><li>Kuriakose, T., Kanneganti, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZBP1:+Innate+sensor+regulating+cell+death+and+inflammation.">ZBP1: Innate sensor regulating cell death and inflammation.</a> Trends in Immunology. 39 (2), 123-134 (2018).</li><li>Maelfait, J., Liverpool, L., Rehwinkel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+sensors+and+programmed+cell+death.">Nucleic acid sensors and programmed cell death.</a> Journal of Molecular Biology. 432 (2), 552-568 (2020).</li><li>Verdonck, S., Nemegeer, J., Vandenabeele, P., Maelfait, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+manipulation+of+host+cell+necroptosis+and+pyroptosis.">Viral manipulation of host cell necroptosis and pyroptosis.</a> Trends in Microbiology. 30 (6), 593-605 (2022).</li><li>Guo, H., 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=Herpes+simplex+virus+suppresses+necroptosis+in+human+cells.">Herpes simplex virus suppresses necroptosis in human cells.</a> Cell Host &amp; Microbe. 17 (2), 243-251 (2015).</li><li>Yu, X., 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=Herpes+simplex+virus+1+(HSV-1)+and+HSV-2+mediate+species-specific+modulations+of+programmed+necrosis+through+the+viral+ribonucleotide+reductase+large+subunit+R1.">Herpes simplex virus 1 (HSV-1) and HSV-2 mediate species-specific modulations of programmed necrosis through the viral ribonucleotide reductase large subunit R1.</a> Journal of Virology. 90 (2), 1088-1095 (2016).</li><li>Huang, Z., 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=RIP1/RIP3+binding+to+HSV-1+ICP6+initiates+necroptosis+to+restrict+virus+propagation+in+mice.">RIP1/RIP3 binding to HSV-1 ICP6 initiates necroptosis to restrict virus propagation in mice.</a> Cell Host &amp; Microbe. 17 (2), 229-242 (2015).</li><li>Guo, H., 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=Species-independent+contribution+of+ZBP1/DAI/DLM-1-triggered+necroptosis+in+host+defense+against+HSV1.">Species-independent contribution of ZBP1/DAI/DLM-1-triggered necroptosis in host defense against HSV1.</a> Cell Death &amp; Disease. 9 (8), 816 (2018).</li><li>Devos, 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=Sensing+of+endogenous+nucleic+acids+by+ZBP1+induces+keratinocyte+necroptosis+and+skin+inflammation.">Sensing of endogenous nucleic acids by ZBP1 induces keratinocyte necroptosis and skin inflammation.</a> The Journal of Experimental Medicine. 217 (7), 20191913 (2020).</li><li>Jiao, H., 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=Z-nucleic-acid+sensing+triggers+ZBP1-dependent+necroptosis+and+inflammation.">Z-nucleic-acid sensing triggers ZBP1-dependent necroptosis and inflammation.</a> Nature. 580 (7803), 391-395 (2020).</li><li>de Reuver, 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=ADAR1+prevents+autoinflammation+by+suppressing+spontaneous+ZBP1+activation.">ADAR1 prevents autoinflammation by suppressing spontaneous ZBP1 activation.</a> Nature. 607 (7920), 784-789 (2022).</li><li>Jiao, H., 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=ADAR1+averts+fatal+type+I+interferon+induction+by+ZBP1.">ADAR1 averts fatal type I interferon induction by ZBP1.</a> Nature. 607 (7920), 776-783 (2022).</li><li>Hubbard, N. W., 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=ADAR1+mutation+causes+ZBP1-dependent+immunopathology.">ADAR1 mutation causes ZBP1-dependent immunopathology.</a> Nature. 607 (7920), 769-775 (2022).</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=ADAR1+masks+the+cancer+immunotherapeutic+promise+of+ZBP1-driven+necroptosis.">ADAR1 masks the cancer immunotherapeutic promise of ZBP1-driven necroptosis.</a> Nature. 606 (7914), 594-602 (2022).</li><li>Adams, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotin+amplification+of+biotin+and+horseradish+peroxidase+signals+in+histochemical+stains.">Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains.</a> The Journal of Histochemistry and Cytochemistry. 40 (10), 1457-1463 (1992).</li><li>Speel, E. J., Hopman, A. H., Komminoth, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyramide+signal+amplification+for+DNA+and+mRNA+in+situ+hybridization.">Tyramide signal amplification for DNA and mRNA in situ hybridization.</a> Methods in Molecular Biology. 326, 33-60 (2006).</li><li>Clutter, M. R., Heffner, G. C., Krutzik, P. O., Sachen, K. L., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyramide+signal+amplification+for+analysis+of+kinase+activity+by+intracellular+flow+cytometry.">Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry.</a> Cytometry A. 77 (11), 1020-1031 (2010).</li><li>Dopie, J., Sweredoski, M. J., Moradian, A., Belmont, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyramide+signal+amplification+mass+spectrometry+(TSA-MS)+ratio+identifies+nuclear+speckle+proteins.">Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins.</a> The Journal of Cell Biology. 219 (9), 201910207 (2020).</li><li>Samson, A. 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</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>Fluorophores</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>Compounds</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30% H2O2</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&#38;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>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>For cell culture: to detach the cells</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>8.0 g/L NaCl</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</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>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>0.2 g/L NaH2PO4</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>0.2 g/L KCl</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</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&#39;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-&#945;</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>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-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 &#176;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>Software</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>Viruses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HSV-1 (mutRHIM) F strain</td>
			<td>produced by&#160; 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&#62;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 ...

Robust visualization of phosphorylated RIPK3 and MLKL, two key signaling events during necroptosis induction, has been technically challenging. The presented tyramide amplification protocol enables sensitive detection of these two molecules. The signal amplification step lowers the detection threshold, allowing robust and reproducible detection of phosphorylated RIPK3 and MLKL.Begin seeding the 90, 000 ZBP1-expressing HT-29 cells in the full strength McCoy's 5A medium in a one centimeter square surface area well plate, allowing high-end microscopy. Use an end volume of 200 microliters per well. Incubate the cells overnight at 37 degrees Celsius with 5%carbon dioxide.Once the cells reach 70 to 80%confluency, infect the cells in 200 microliters preheated, full-strength McCoy's 5A medium containing herpes simplex virus one, encoding a rim mutant ICP6 at a multiplicity of infection of five. Incubate the cells with the virus for nine hours to induce necroptosis. As a positive control for necroptosis induction, stimulate the cells for four hours with 200 microliters of preheated necroptosis-inducing cocktail.As a negative control, add 22 microliters of 10 micromolar GSK840 preheated at 37 degrees Celsius to inhibit RIPK3 kinase activity. Include this inhibitor in an untreated condition and after necroptosis stimulation to prevent the autophosphorylation of serine 227. To fix the cells, remove the medium and wash the cells with 200 microliters of PBS.Then remove the PBS and add 150 microliters of 4%paraformaldehyde equilibrated at room temperature. Incubate the cells at room temperature for 30 minutes. After fixation, remove the 4%paraformaldehyde, and wash the cells three times with 200 microliters of PBS.The samples can be stored in excess of PBS at four degree Celsius overnight until further processing. Remove the PBS from the plate, and incubate the cells with 100 microliters of permeation buffer at room temperature for 30 minutes on a tilting laboratory shaker. After incubation, remove the permeabilization buffer, and wash the cells with 100 microliters of wash buffer.Incubate the imaging chamber with wash buffer for five minutes at room temperature on a tilting laboratory shaker. To prevent the non-specific binding of primary antibodies, incubate the plate with 100 microliters of blocking medium at room temperature for two hours on a tilting laboratory shaker. Next, wash the wells three times with 100 microliters of wash buffer with five minutes of incubation at room temperature on a tilting laboratory shaker.After removing the wash buffer, add 100 microliters of the primary antibodies to the imaging chamber. Ensure to include a no primary antibody condition to visualize the potential background of the tyramide signal amplification, and to set the masking threshold. Incubate the chamber overnight at four degree Celsius.Remove the primary antibody mix, and wash the wells three times with 100 microliters of wash buffer with five minutes of incubation at room temperature on a tilting laboratory shaker. After removing the wash buffer, incubate the cells with 100 microliters of HRP-labeled secondary antibody, recognizing the species of the primary antibody to be amplified for 30 minutes on a tilting laboratory shaker. During antibody incubation, prepare the biotinylated tyramide mix.To trigger the enzymatic activity of HRP, add an oxidizing substrate to the TSA buffer freshly before use. Then dilute the biotinylated tyramide in the prepared TSA buffer using the optimized dilution. After incubation with HRP-labeled secondary antibody, wash the wells three times with wash buffer with five minutes of incubation on a tilting laboratory shaker.After removing the wash buffer from the well, add diluted biotin-tyramide to the well to an end volume of 100 microliters. Incubate the reaction at room temperature for 10 minutes on a tilting laboratory shaker, followed by three wash buffer washes. Prepare the staining mix containing the secondary antibody, coupled streptavidin, and nuclear stain and wash buffer.Remove the wash buffer and incubate the plate with 100 microliters of staining mix at room temperature for one hour on a tilting laboratory shaker. Keep the imaging chambers shielded from light from this step onward. After staining, consecutively wash twice with wash buffer, and rinse the wells two times with PBS.Store the samples in excess of PBS at four degree Celsius to preserve the staining until imaging. The imaging chamber is now ready to be visualized on a confocal microscope. The inclusion of tyramide signal amplification enabled the robust detection of RIPK3 phosphorylated at serine 227 in the cytosol of herpes simplex virus one encoding a rim mutant ICP6 infected cells.A laser power of 2%was sufficient for sensitive detection. In standard indirect immunofluorescence, a higher laser power remained insufficient for the detection of phospho-RIPK3. In quantification of the three-dimensional Z-stack images, the infected cells showed an approximately 20-fold increase of voxels positive for phosphorylated RIPK3 over mock-treated cells.Increased phospho-RIPK3 staining of wild-type herpes simplex virus one infected cells compared to mock-treated cells indicated that the rim domain of ICP6 is unable to block RIPK3 phosphorylation at serine 227 completely. GSK840 binds to the kinase domain of RIPK3, and prevents RIPK3 phosphorylation at serine 227 upon ZBP1 activation, confirming the specificity of the tyramide signal amplification-mediated phospho-RIPK3 detection method. The mock-treated cells showed low in slightly punctuated cytosolic staining of MLKL phosphorylation at serine 358, while strong staining was detected in the cytosol, nucleus, and plasma membrane of the infected cells.The standard indirect immunofluorescence required 40%laser power for the specific detection of a phospho-MLKL signal in the infected cells. In contrast, tyramide signal amplification increased detection sensitivity of phospho-MLKL, thereby lowering the necessary laser power to 6%3D Z-stack image quantification showed an approximately 90-fold increase in the number of voxels positive for phospho-MLKL using tyramide signal amplification, compared to a seven-fold increase using the standard method, indicating improved detection threshold and sensitivity for phospho-MLKL. Similar to herpes simplex virus one, influenza A virus infection triggered ZBP1-dependent necroptosis.Tyramide signal amplification allowed the robust detection of phospho-RIPK3 and phospho-MLKL, indicating necroptosis. To interpret the confocal staining, several staining controls need to be included. This entails a no primary condition, single stains, as well as a positive control.By adapting this protocol, multiple targets can be amplified using sequential TSA. This would allow the detection of phosphorylated RIPK3 and MLKL within the same cell. Sensitive detection of necroptosis biomarkers can be insightful for ongoing ZBP1 research on autoinflammation or virus infection in which the executed ZBP1-dependent cell death pathways still need to be confirmed as either necroptosis, apoptosis, or pyroptosis.

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

Research

JoVE Journal

Immunology and Infection

2.0K Views.  VIB-UGent Center for Inflammation Research. Robust visualization of phosphorylated RIPK3 and MLKL, two key signaling events during necroptosis induction, has been technically challenging. The presented tyramide amplification protocol enables sensitive detection of these two molecules. The signal amplification step lowers the detection threshold, allowing robust and reproducible detection of phosphorylated RIPK3 and MLKL.Begin seeding the 90, 000 ZBP1-expressing HT-29 cells in the full strength McCoy's 5A medium in a one centimet...

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