The authors thank the department of Pathology of Hopital Europ&#233;en Georges Pompidou and Necker (Laurianne Chambolle, Elodie Michel, and Gis&#232;le Legall); the Histology platform of PARCC, Hopital Europ&#233;en Georges Pompidou (Corinne Lesaffre); Virginia Clark for language editing; Alexandra Elbakyan for her contribution.

The protocol follows ethical guidelines and was approved by the Ethical Committee (Comit&#233;-de-Protection-des-Personnes Ile-de-France-II, #2015-09-04).
1. Preparation of the materials
<ol>
	<li>Preparation of 1x wash buffer
	<ol>
		<li>Prepare 3 L of 1x wash buffer by adding 2.94 L of distilled water and one bottle (60 mL) of wash buffer (50x) (see the Table of Materials) to a large carboy. Mix well. 
		NOTE: The 1x wash buffer may be prepared ahead of time and stored at room temperature for up to 1 month.</li>
	</ol>
	</li>
	<li>Preparation of counterstaining reagents
	<ol>
		<li>Prepare 50% hematoxylin.
		<ol>
			<li>In a fume hood, add 100 mL of Gill&#8217;s hematoxylin I (see the Table of Materials) to 100 mL of distilled water in a staining dish. 
			NOTE: The 50% hematoxylin staining solution can be reused for up to 1 week.</li>
		</ol>
		</li>
		<li>Prepare 0.02% (w/v) ammonia water (bluing reagent).</li>
		<li>In the fume hood, add 1.43 mL of 1 N ammonium hydroxide to 250 mL of distilled water in a graduated cylinder or another container. Seal the cylinder with paraffin film. Mix its contents well for 3x&#8211;5x. 
		NOTE: For assay quantitation, it is critical to use ammonium hydroxide. The reagents may be prepared ahead of time. Ensure all containers remain covered.</li>
	</ol>
	</li>
	<li>Preparation of 1x target retrieval reagent
	<ol>
		<li>In a large beaker, add 70 mL of 10x target retrieval reagent (see the Table of Materials) to 630 mL of distilled water.</li>
		<li>Place the beaker on a heating plate with a magnetic stirrer. Cover it with aluminum foil.</li>
		<li>Boil its contents at 100 &#176;C for 10&#8211;15 min. 
		NOTE: Do not let it boil for more than 30 min.</li>
	</ol>
	</li>
	<li>Reagent equilibration
	<ol>
		<li>Ensure the hybridization oven is on and at 40 &#176;C. Place wet humidifying paper at the bottom of the tray. 
		NOTE: A hybridization oven (see the Table of Materials) is needed for steps 3.4 to 4.2.</li>
		<li>Remove the amplification reagents (AMP1&#8211;AMP6, see the Table of Materials) from the refrigerator and keep them at room temperature, at least 30 min before the relevant incubation step.</li>
		<li>Before each use, warm the target and/or control probes for at least 10 min at 40 &#176;C in the oven or in a water bath or incubator.</li>
	</ol>
	</li>
</ol>
2. Adhesion enhancing and deparaffinization in the fume hood
NOTE: Start the protocol with 3&#8211;5 &#181;m-thick histological samples mounted on unstained slides.
<ol>
	<li>In order to enhance adhesion, bake the slides at 60 &#176;C for 1 h or at 40 &#176;C overnight in an oven.</li>
	<li>Charge the slide rack with unstained histological slides. Immerse the slide rack for 5 min in fresh xylene contained in a staining dish, with occasional agitation. repeat with fresh xylene.</li>
	<li>Immerse the slide rack for 3 min in fresh 100% ethanol contained in a staining dish, with constant agitation. Repeat with fresh 100% ethanol.</li>
	<li>Let the slides dry for 2 min at room temperature. 
	NOTE: Do not reuse deparaffinization reagents for dehydration of the slides after the assay.</li>
</ol>
3. Tissue pretreatment
NOTE: These steps follow the &#8220;standard&#8221; pretreatment recommendation according to the manufacturer&#8217;s instructions for head and neck samples. The timing of sections 3.1 and 3.2 may need to be adjusted depending on the manipulated tissue.
<ol>
	<li>Blockade of peroxidase activity
	<ol>
		<li>Add 4&#8211;6 drops of hydrogen peroxide (see the Table of Materials) to each slide and incubate them for 10 min at room temperature.</li>
		<li>Wash the slides 2x for 2 min in distilled water at room temperature.</li>
	</ol>
	</li>
	<li>Breakage of RNA/tissue bounds 
	<ol>
		<li>With a claw, remove the aluminum foil from the boiling 1x target retrieval reagent (TTR1x) from section 1.3 and stop stirring. Immerse the slide rack slowly and very carefully for 15 min. Cover the beaker again with the aluminum foil. 
		NOTE: Simmering has to persist during this step. 
		CAUTION: Use the claw to manipulate the aluminum foil and the slide rack so as to avoid burn injuries. Make sure to wear proper personal protective equipment, such as gloves and a lab coat.</li>
		<li>With the claw, immediately transfer the hot slide rack to a distilled water bath and wash it for 2 min. 
		NOTE: Make sure the samples do not cool down in the TTR1x.</li>
		<li>Wash the slides in fresh 100% ethanol for 2 min.</li>
		<li>Let the slides dry at room temperature for 2 min.</li>
	</ol>
	</li>
	<li>Barrier creation
	<ol>
		<li>With a hydrophobic barrier pen (see the Table of Materials), draw a barrier around the sample. Let it dry out at for least 5 min. 
		NOTE: Allow the barrier to really dry out. If it wears out during the procedure, do not hesitate to draw it again. Avoid touching the tissue with the pen. The protocol can be paused overnight here.</li>
	</ol>
	</li>
	<li>Protease digestion
	<ol>
		<li>Place the slides on the slide rack and add ~4 drops of Protease Plus per sample (see the Table of Materials).</li>
		<li>Cover the humidity control tray with a lid and insert it into the hybridization oven for 30 min at 40 &#176;C. 
		NOTE: To prevent evaporation, make sure the turn knob is completely turned to the lock position.</li>
		<li>Remove the tray from the oven and remove the slide rack.</li>
		<li>One slide at a time, quickly remove any excess liquid and place the slide in a slide rack submerged in a staining dish filled with distilled water.</li>
		<li>Wash the slides 2x for 2 min in distilled water at room temperature. Agitate constantly.</li>
	</ol>
	</li>
</ol>
4. Running the assay
NOTE: Do not let sections dry out between the incubation steps.
<ol>
	<li>Hybridization of the HPV probe 
	NOTE: Ensure the probes are prewarmed to dissolve any precipitation prior to use. For this step, instead of HPV probe, peptidyl-prolyl isomerase B (PPIB) for positive control or dihydrodipicolinate reductase (DAPB) for negative control (see the Table of Materials) may also be used.
	<ol>
		<li>Tap and/or flick the slides to remove any excess liquid and place them in the slide rack. Add ~4 drops of HPV probe to entirely cover each section.</li>
		<li>Cover the tray with a lid and insert it into the oven for 2 h at 40 &#176;C. 
		NOTE: To prevent evaporation, make sure the turn nob is completely turned to the lock position.</li>
		<li>Remove the tray from the oven and remove the slide rack.</li>
		<li>One slide at a time, quickly remove any excess liquid and place the slide in a slide rack submerged in a staining dish filled with 1x wash buffer.</li>
		<li>Wash the slides in 1x wash buffer for 2 min at room temperature with constant agitation. Repeat this with fresh 1x wash buffer.</li>
	</ol>
	</li>
	<li>Hybridization of AMP1, AMP2, AMP3, and AMP4 
	NOTE: These steps include hybridization in the hybridization oven and AMP1&#8211;AMP4 from the purchased kit (see the Table of Materials).
	<ol>
		<li>Tap and/or flick to remove any excess liquid from the slides and place them in the slide rack. Add ~4 drops of AMP1 to entirely cover each section.</li>
		<li>Cover the tray with a lid and insert it into the oven for 30 min at 40 &#176;C.</li>
		<li>Remove the tray from the oven and remove the slide rack.</li>
		<li>One slide at a time, quickly remove any excess liquid and place the slide in a slide rack submerged in a staining dish filled with 1x wash buffer.</li>
		<li>Wash the slides in 1x wash buffer for 2 min at room temperature with constant agitation. Repeat this with fresh 1x wash buffer.</li>
		<li>Repeat steps 4.2.1&#8211;4.2.5, but use ~4 drops of AMP2 instead of AMP1 and incubate for 15 min at 40 &#176;C. Wash the slides 2x for 2 min, both times in fresh wash buffer.</li>
		<li>Repeat steps 4.2.1&#8211;4.2.5, but use ~4 drops of AMP3 instead of AMP1 and incubate for 30 min at 40 &#176;C. Wash the slides 2x for 2 min, both times in fresh wash buffer.</li>
		<li>Repeat steps 4.2.1&#8211;4.2.5, but use ~4 drops of AMP4 instead of AMP1 and incubate for 15 min at 40 &#176;C. Wash the slides 2x for 2 min, both times in fresh wash buffer.</li>
	</ol>
	</li>
	<li>Hybridization of AMP5 and AMP6 
	NOTE: These steps do not include hybridization in the oven but hybridization at room temperature. AMP5 and AMP6 come from the purchased kit (see the Table of Materials).
	<ol>
		<li>Tap and/or flick the slides to remove any excess liquid and place them in the slide rack. Add ~4 drops of AMP5 to entirely cover each section.</li>
		<li>Cover the tray with a lid and incubate for 30 min at room temperature.</li>
		<li>One slide at a time, quickly remove any excess liquid and place it in a slide rack submerged in a staining dish filled with 1x wash buffer.</li>
		<li>Wash the slides in 1x wash buffer for 2 min at room temperature with constant agitation. Repeat this with fresh 1x wash buffer.</li>
		<li>Repeat steps 4.3.1&#8211;4.3.4, but use ~4 drops of AMP6 instead of AMP5 and incubate for 15 min at room temperature. Wash the slides 2x for 2 min, both times in fresh wash buffer.</li>
	</ol>
	</li>
</ol>
5. Signal detection with 3,3&#39;-diaminobenzidine
CAUTION: Diaminobenzidine (DAB) is toxic. Follow appropriate precautions and safety guidelines when disposing of and handling this chemical.
<ol>
	<li>Mix equal volumes of DAB-A and DAB-B (see the Table of Materials) in an appropriately sized tube by dispensing the same number of drops of each solution. Make ~120 &#181;L of DAB substrate per section (~2 drops of each reagent/total of 4). Mix it well for 3x&#8211;5x.</li>
	<li>Take each slide, one at a time, from the slide rack and tap and/or flick to remove the excess liquid before placing it in the slide rack.</li>
	<li>Pipette ~120 &#956;L of DAB onto each tissue section. Ensure the sections are covered, and incubate for 10 min at room temperature.</li>
	<li>Dispose the remaining DAB according to local regulation and insert the slide into a slide rack submerged in a staining dish filled with tap water.</li>
</ol>
6. Counterstaining
<ol>
	<li>Move the slide rack to a staining dish containing 50% hematoxylin staining solution let it rest for 30 s at room temperature. Note that the slides will become purple.</li>
	<li>Immediately transfer the slide rack back to a staining dish containing tap water, and wash the slides 3x&#8211;5x by moving the rack up and down.</li>
	<li>Keep repeating the washing step with fresh tap water until the slides are clear, while sections remain purple.</li>
	<li>Replace the tap water in the staining dish with 0.02% ammonia water. Move the rack up and down 2x&#8211;3x. Note that the tissue section should turn blue.</li>
	<li>Replace the ammonia water with tap water. Wash the slides 3x&#8211;5x.</li>
</ol>
7. Dehydration
<ol>
	<li>Move the slide rack to a staining dish containing 70% ethanol in the fume hood and let it rest for 2 min with occasional agitation.</li>
	<li>Move the slide rack to a first staining dish containing 100% ethanol and let it rest for 2 min with occasional agitation.</li>
	<li>Move the slide rack to a second staining dish containing 100% ethanol and let it rest for 2 min with occasional agitation.</li>
	<li>Move the slide rack to a staining dish containing xylene and let it rest for 5 min with occasional agitation.</li>
</ol>
8. Slide mounting
<ol>
	<li>Remove the slides from the slide rack and lay them flat with the sections facing up in the fume hood.</li>
	<li>Mount one slide at a time by adding 1 drop of a xylene-based mounting medium to each slide and carefully placing a 24 mm x 50 mm coverslip over the section. Avoid trapping any air bubbles.</li>
	<li>Air-dry the slides for &#8805;5 min.</li>
</ol>
9. Sample evaluation
<ol>
	<li>Examine the tissue sections under a standard brightfield microscope at 20x&#8211;40x magnification.</li>
</ol>

<ol>
	<li>Lowy, D. R., Schiller, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+HPV-associated+cancer+globally.">Reducing HPV-associated cancer globally.</a> Cancer Prevention Research.(Philadelphia, PA). 5 (1), 18-23 (2012).</li><li>Laban, S., Hoffmann, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Papillomavirus+Immunity+in+Oropharyngeal+Cancer:+Time+to+Change+the+Game?.">Human Papillomavirus Immunity in Oropharyngeal Cancer: Time to Change the Game?.</a> Clinical Cancer Research. 24 (3), 505-507 (2018).</li><li>Wiest, T., Schwarz, E., Enders, C., Flechtenmacher, C., Bosch, F. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+intact+HPV16+E6/E7+gene+expression+in+head+and+neck+cancers+with+unaltered+p53+status+and+perturbed+pRb+cell+cycle+control.">Involvement of intact HPV16 E6/E7 gene expression in head and neck cancers with unaltered p53 status and perturbed pRb cell cycle control.</a> Oncogene. 21 (10), 1510-1517 (2002).</li><li>Ndiaye, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPV+DNA,+E6/E7+mRNA,+and+p16INK4a+detection+in+head+and+neck+cancers:+a+systematic+review+and+meta-analysis.">HPV DNA, E6/E7 mRNA, and p16INK4a detection in head and neck cancers: a systematic review and meta-analysis.</a> The Lancet Oncology. 15 (12), 1319-1331 (2014).</li><li>Mills, A. M., Dirks, D. C., Poulter, M. D., Mills, S. E., Stoler, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HR-HPV+E6/E7+mRNA+In+Situ+Hybridization.">HR-HPV E6/E7 mRNA In Situ Hybridization.</a> The American Journal of Surgical Pathology. 41 (5), 607-615 (2017).</li><li>Mendez-Pena, J. E., Sadow, P. M., Nose, V., Hoang, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+chromogenic+in+situ+hybridization+assay+with+clinical+automated+platform+is+a+sensitive+method+in+detecting+high-risk+human+papillomavirus+in+squamous+cell+carcinoma.">RNA chromogenic in situ hybridization assay with clinical automated platform is a sensitive method in detecting high-risk human papillomavirus in squamous cell carcinoma.</a> Human Pathology. 63, 184-189 (2017).</li><li>Mirghani, 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=Diagnosis+of+HPV+driven+oropharyngeal+cancers:+Comparing+p16+based+algorithms+with+the+RNAscope+HPV-test.">Diagnosis of HPV driven oropharyngeal cancers: Comparing p16 based algorithms with the RNAscope HPV-test.</a> Oral Oncology. 62, 101-108 (2016).</li><li>Lewis, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Papillomavirus+Testing+in+Head+and+Neck+Carcinomas:+Guideline+From+the+College+of+American+Pathologists.">Human Papillomavirus Testing in Head and Neck Carcinomas: Guideline From the College of American Pathologists.</a> Archives of Pathology &amp; Laboratory Medicine. 142 (5), 559-597 (2018).</li><li>Mills, A. M., Coppock, J. D., Willis, B. C., Stoler, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPV+E6/E7+mRNA+In+Situ+Hybridization+in+the+Diagnosis+of+Cervical+Low-grade+Squamous+Intraepithelial+Lesions+(LSIL).">HPV E6/E7 mRNA In Situ Hybridization in the Diagnosis of Cervical Low-grade Squamous Intraepithelial Lesions (LSIL).</a> The American Journal of Surgical Pathology. 42 (2), 192-200 (2018).</li><li>El-Naggar, A., Chan, J. K. C., Grandis, J. R., Takata, T., Slootweg, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> WHO Classification of Head and Neck Tumours. , (2017).</li><li>Ang, K. K., 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+papillomavirus+and+survival+of+patients+with+oropharyngeal+cancer.">Human papillomavirus and survival of patients with oropharyngeal cancer.</a> The New England Journal of Medicine. 363 (1), 24-35 (2010).</li><li>Badoual, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-1-expressing+tumor-infiltrating+T+cells+are+a+favorable+prognostic+biomarker+in+HPV-associated+head+and+neck+cancer.">PD-1-expressing tumor-infiltrating T cells are a favorable prognostic biomarker in HPV-associated head and neck cancer.</a> Cancer Research. 73 (1), 128-138 (2013).</li><li>Outh-Gauer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunotherapy+in+head+and+neck+cancers:+a+new+challenge+for+immunologists,+pathologists+and+clinicians.">Immunotherapy in head and neck cancers: a new challenge for immunologists, pathologists and clinicians.</a> Cancer Treatment Reviews. 65, 54-64 (2018).</li><li>Mirghani, 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=Diagnosis+of+HPV-driven+head+and+neck+cancer+with+a+single+test+in+routine+clinical+practice.">Diagnosis of HPV-driven head and neck cancer with a single test in routine clinical practice.</a> Modern Pathology. 28 (12), 1518-1527 (2015).</li><li>Bishop, J. A., 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=Detection+of+Transcriptionally+Active+High-risk+HPV+in+Patients+With+Head+and+Neck+Squamous+Cell+Carcinoma+as+Visualized+by+a+Novel+E6/E7+mRNA+In+Situ+Hybridization+Method.">Detection of Transcriptionally Active High-risk HPV in Patients With Head and Neck Squamous Cell Carcinoma as Visualized by a Novel E6/E7 mRNA In Situ Hybridization Method.</a> The American Journal of Surgical Pathology. 36 (12), 1874-1882 (2012).</li><li>Hsieh, M. -. S., Lee, Y. -. H., Jin, Y. -. T., Huang, W. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strong+SOX10+expression+in+human+papillomavirus-related+multiphenotypic+sinonasal+carcinoma:+report+of+6+new+cases+validated+by+high-risk+human+papillomavirus+mRNA+in+situ+hybridization+test.">Strong SOX10 expression in human papillomavirus-related multiphenotypic sinonasal carcinoma: report of 6 new cases validated by high-risk human papillomavirus mRNA in situ hybridization test.</a> Human Pathology. 82, 264-272 (2018).</li><li>Shi, 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=Comparative+Prognostic+Value+of+HPV16+E6+mRNA+Compared+With+In+Situ+Hybridization+for+Human+Oropharyngeal+Squamous+Carcinoma.">Comparative Prognostic Value of HPV16 E6 mRNA Compared With In Situ Hybridization for Human Oropharyngeal Squamous Carcinoma.</a> Journal of Clinical Oncology. 27 (36), 6213-6221 (2009).</li><li>Kuo, K. -. 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=The+biomarkers+of+human+papillomavirus+infection+in+tonsillar+squamous+cell+carcinoma&#8212;molecular+basis+and+predicting+favorable+outcome.">The biomarkers of human papillomavirus infection in tonsillar squamous cell carcinoma&#8212;molecular basis and predicting favorable outcome.</a> Modern Pathology. 21 (4), 376-386 (2008).</li><li>Augustin, J., 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=Evaluation+of+the+efficacy+of+the+four+tests+(p16+immunochemistry,+PCR,+DNA+and+RNA+In+situ+Hybridization)+to+evaluate+a+Human+Papillomavirus+infection+in+head+and+neck+cancers:+a+cohort+of+348+French+squamous+cell+carcinomas.">Evaluation of the efficacy of the four tests (p16 immunochemistry, PCR, DNA and RNA In situ Hybridization) to evaluate a Human Papillomavirus infection in head and neck cancers: a cohort of 348 French squamous cell carcinomas.</a> Human Pathology. , (2018).</li><li>Jung, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+and+clinical+relevance+of+transcriptionally+active+human+papillomavirus+(HPV)+infection+in+oropharynx+squamous+cell+carcinoma.">Biological and clinical relevance of transcriptionally active human papillomavirus (HPV) infection in oropharynx squamous cell carcinoma.</a> International Journal of Cancer. 126 (8), 1882-1894 (2009).</li><li>Augustin, J., 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=HPV+RNA+CISH+score+identifies+two+prognostic+groups+in+a+p16+positive+oropharyngeal+squamous+cell+carcinoma+population.">HPV RNA CISH score identifies two prognostic groups in a p16 positive oropharyngeal squamous cell carcinoma population.</a> Modern Pathology. , (2018).</li><li>Evans, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPV+E6/E7+RNA+In+Situ+Hybridization+Signal+Patterns+as+Biomarkers+of+Three-Tier+Cervical+Intraepithelial+Neoplasia+Grade.">HPV E6/E7 RNA In Situ Hybridization Signal Patterns as Biomarkers of Three-Tier Cervical Intraepithelial Neoplasia Grade.</a> PLOS ONE. 9 (3), e91142 (2014).</li><li>Dreyer, J. H., Hauck, F., Oliveira-Silva, M., Barros, M. H. M., Niedobitek, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+HPV+infection+in+head+and+neck+squamous+cell+carcinoma:+a+practical+proposal.">Detection of HPV infection in head and neck squamous cell carcinoma: a practical proposal.</a> Virchows Archiv. 462 (4), 381-389 (2013).</li><li>Bingham, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAscope+in+situ+hybridization+confirms+mRNA+integrity+in+formalin-fixed,+paraffin-embedded+cancer+tissue+samples.">RNAscope in situ hybridization confirms mRNA integrity in formalin-fixed, paraffin-embedded cancer tissue samples.</a> Oncotarget. , (2017).</li></ol>

CB: boards, seminars: MSD, BMS, Astrazeneca, Roche

HPV RNA CISH performed with a purchased kit is a powerful tool for the detection of viral transcripts and it indicates active HPV infection. Performed manually, the steps of the protocol are overall easy to follow, and the purchased kit is convenient. This technique allows the staining of 19 histological samples plus one control slide at once, and the assay lasts around 8 h. It is critical not to let the samples dry out between steps unless otherwise mentioned. The pretreatment condition may need to be adjusted depending...

HPV RNA CISH is a powerful tool for the detection of active HPV infection, which may prove crucial in benign or malignant lesions in various locations such as the oropharynx or the uterine cervix. The detection of an active HPV infection may support the diagnosis of an HPV-induced lesion and, thereby, influence its treatment and prognosis.
HPV is the most frequent sexually transmitted infection, and over 100 viral genotypes have been described1. Schematically, low-risk genotypes such as genotypes 6 and 11 are known to induce genital warts, recurrent respiratory papillomatosis, and other benign lesions, whereas high-risk genotypes such as genotypes 16 and also 18 are responsible for most cervical cancers and anal cancers and play a role in HNSCC oncogenesis in variable proportions as accounted for by regional epidemiological data2.
Several tools are available for the detection of HPV infection. As a high-risk HPV infection leads to the expression of viral oncogenic proteins E6 and E73, the detection of E6 and E7 transcripts is widely viewed as the gold standard for active HPV infection identification4. HPV RNA CISH can be performed on FFPE samples that are quite easily obtained from patients suffering from various HPV-related diseases. Its performance has been evaluated in squamous intraepithelial neoplasia in the cervix, the anus, and the vagina, and in invasive squamous cell carcinoma in the cervix, the anus, and the upper aerodigestive tract5: it achieves a sensitivity of over 98% among HPV DNA polymerase chain reaction (PCR)-positive cases. This is slightly better than p16 immunostaining (93%) and HPV DNA in situ hybridization (DNA ISH: 97%), which are more commonly used. In another cohort of 57 patients with squamous cell carcinoma (SCC) arising from the head and neck region, the genital region, the skin, and the urinary tract, compared to HPV DNA ISH, HPV RNA CISH achieved better sensitivity (100% versus 88%) and specificity (87% versus 74%)6.
P16 immunostaining is an indirect marker reflecting cell cycle disruption that may be caused (but not exclusively) by HPV infection4,7. This cost-effective test possesses good sensitivity and a negative predictive value and is recommended as a surrogate marker of high-risk HPV infection in oropharynx cancer (OPC) by the College of American Pathologists (CAP) and by the Union for International Cancer Control (UICC)8.
Though this paper solely focuses on the detection of HPV in HNSCC, HPV RNA CISH is clinically relevant in various other conditions that involve HPV infection. For instance, this technique may improve the accuracy of the diagnosis of low-grade squamous intraepithelial lesions of the cervix (LSIL, formerly known as cervical intraepithelial neoplasia, grade 1 [CIN1]) for morphologically ambiguous cases9. Regarding oropharyngeal SCC, HPV RNA CISH allows the identification of HPV-related SCC, labeled as distinct from HPV-unrelated oropharyngeal SCC in the recent eighth edition of the TNM Classification of Head and Neck Cancer (of the Union for International Cancer Control [UICC])10. Since HPV-related SCC exhibits a better prognosis with longer survival and enhanced radiotherapy and chemotherapy sensitivity than HPV-unrelated SCC11,12,13, the detection of HPV infection may impact patient management14,15. Besides, HPV RNA CISH can be used for the diagnosis of HPV-related multiphenotypic sinonasal carcinoma with a higher signal than HPV DNA CISH16. Several multivariate analyses suggest that the detection of E6 and E7 transcripts is correlated with a better prognosis in oropharyngeal SCC overall7,15,17,18 and in the subgroup of p16-positive oropharyngeal SCC19,20.
Here we present the protocol for manual HPV RNA CISH performed on FFPE slides with a kit obtained from the manufacturer.&#160;

<table><tbody><tr><td>Hematoxylin solution, Gill No. 1</td><td>Merck</td><td>GHS132</td><td></td></tr><tr><td>HybEZ Oven (110v)</td><td>Advanced Cell Diagnostics Inc.</td><td>321710</td><td></td></tr><tr><td>HybEZ slide rack</td><td>Advanced Cell Diagnostics Inc.</td><td>300104</td><td></td></tr><tr><td>ImmEdge Hydrophobic Barrier Pen</td><td>Advanced Cell Diagnostics Inc.</td><td>310018</td><td></td></tr><tr><td>RNAscope 2.5 HD Detection Reagents-BROWN</td><td>Advanced Cell Diagnostics Inc.</td><td>322310</td><td>This kit includes amplification reagents AMP1, AMP2, AMP3, AMP4, AMP5 and AMP6, and detection reagents DAB-A and DAB-B</td></tr><tr><td>RNAscope 3-Plex Negative Control Probe</td><td>Advanced Cell Diagnostics Inc.</td><td>320871</td><td>DAPB</td></tr><tr><td>RNAscope 3-Plex Positive Control Probe</td><td>Advanced Cell Diagnostics Inc.</td><td>320861</td><td>PPIB</td></tr><tr><td>RNAscope H202 &amp;amp; Protease Plus Reagent</td><td>Advanced Cell Diagnostics Inc.</td><td>322330</td><td>Hydrogen Peroxyde x2 and Protease Plus x 1</td></tr><tr><td>RNAscope Probe- HPV16/18</td><td>Advanced Cell Diagnostics Inc.</td><td>311121</td><td></td></tr><tr><td>RNAscope Target Retrieval Reagents</td><td>Advanced Cell Diagnostics Inc.</td><td>322000</td><td></td></tr><tr><td>RNAscope Wash Buffer Reagents</td><td>Advanced Cell Diagnostics Inc.</td><td>310091</td><td>Wash Buffer 50X x4</td></tr></tbody></table>

chromogenic in situ hybridization as a tool for hpv-related head and neck cancer diagnosis

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated with a better outcome than alcohol- and tobacco-related OPSCC. Chromogenic in situ hybridization (CISH) of HPV viral RNA could allow the semiquantitative evaluation of viral transcripts of the oncogenic proteins E6 and E7 and an in situ visualization with a good spatial resolution. This technique allows the diagnosis of an active infection with the visualization of HPV transcription in the tumoral HPV-infected cells. An advantage of this technique is the avoidance of contamination from nonneoplastic HPV-infected cells adjacent to the tumor. Overall, its good diagnosis performances have it considered to be the gold standard for active HPV infection identification. Since E6 and E7 viral protein interaction with cell proteins pRb and p53 is mandatory for cell transformation, HPV RNA CISH is functionally relevant and acutely reflects active oncogenic HPV infection. This technique is clinically relevant as well since &#34;low&#34; or &#34;high&#34; HPV transcription levels helped the identification of two prognosis groups among HPV-related p16-positive head and neck cancer patients. Here we present the protocol for manual HPV RNA CISH performed on formalin-fixed paraffin-embedded (FFPE) slides with a kit obtained from the manufacturer. Instead of chromogenic revelation, RNA in situ hybridization may also be performed with fluorescent revelation (RNA FISH). It may also be combined with conventional immunostaining.

As described here, in head and neck squamous cell cancer, a case may be considered positive in the presence of brown punctiform staining in the cytoplasm or in the nuclei of tumor cells. In most studies, the signal is considered as either “positive” or “not detected”14. Methods of semiquantification of the signal have been reported but lack standardization between teams. For example, in some studies, the signals were scored as 1+ with 1–3 dots per tumor cell, 2+ with 4–9 dots per tumor...

Watch this Scientific Journal Video about Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis at JoVE.com

Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis

Human papillomavirus (HPV) RNA chromogenic in situ hybridization is considered to be one of the gold standards for active human papillomavirus infection detection within tumors. It allows the visualization of HPV E6-E7 mRNA expression with localization and semiquantitative evaluation of its signal.

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated ...

chromogenic-situ-hybridization-as-tool-for-hpv-related-head-neck

Research

JoVE Journal

Genetics

10.3K Views.  Paris Descartes University. HPV RNA CISH maakt de detectie van een actieve infectie van het humaan papillomavirus mogelijk. Aangezien de in-situ hybridisatie doelen HPV RNA deze techniek maakt de visualisatie van een actieve transcriptie van het virus. Het heeft een nauwkeurige ruimtelijke resolutie en goede diagnose prestaties.De detectie van HPV kan de diagnose van verschillende HPV-gerelateerde laesies ondersteunen, zoals orofaryngeale plaveiselcelcarcinoom of cervicale neoplasie. Het kan ook invloed hebben op...

Video: Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Cancer Research

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current &#39;gold standard&#39; method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The presence of high-risk HPV in head and neck tumor tissue is associated with favorable outcomes. The recently developed RNA in situ hybridization technique called RNAscope allows direct visualization of HPV E6/E7 mRNA in FFPE tissue sections.

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection ...

Medicine

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

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Biology

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and MET&#916;14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors &#945; and &#946; in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust ...

HCR-DNA FISH: A Fluorescence In Situ Hybridization Technique to Detect Viral DNA in Infected Cells

Source: Wei Liu&#160;et al., <a href="https://www.jove.com/v/58950"> Merkel Cell Polyomavirus Infection and Detection</a>, J. Vis. Exp. (2019).
In this video, the cells infected with MCPyV virions were subjected to in situ hybridization chain reaction to detect MCPyV specific genomes. Further, the DNA-HCR technique was combined with FISH to visualize the MCPyV DNA in infected human skin cells.

Source: Wei Liu&#160;et al.,  Merkel Cell Polyomavirus Infection and Detection, J. Vis. Exp. (2019).
In this video, the cells infected with MCPyV virions ...

JoVE Encyclopedia of Experiments

Skin Cancer

Assays and techniques

FFPE Tissue Pretreatment for RNA CISH: A Procedure to Process Formalin-Fixed Paraffin-Embedded Tissue Sections for RNA Chromogenic In Situ Hybridization

Source: Outh-Gauer, S. et al. <a href="https://www.jove.com/video/59422" target="_blank">Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis.</a> J. Vis. Exp. (2019)
This video demonstrates the steps involved in the pretreatment of FFPE tissue specimens for RNA chromogenic in situ hybridization. The pretreatment process helps maintain nucleic acid integrity within the cells and overall tissue morphology.

Source: Outh-Gauer, S. et al. Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis. J. Vis. Exp. (2019)
This video ...

Head and Neck Cancer

Assays & techniques

Probe Hybridization and Signal Amplification in RNA In Situ Hybridization: A Technique for Detecting Specific RNA Sequences in Tissue Sections

Source: Outh-Gauer, S. et al. <a href="https://www.jove.com/video/59422" target="_blank">Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis.</a> J. Vis. Exp. (2019)
This video describes a sequential hybridization strategy that involves hybridizing mRNA probes to specific target mRNA. Following probe hybridization, signal amplification is performed to enhance resolution via reduction of signal-to-noise ratio during RNA-CISH of histological samples.

RNA CISH Signal Detection: A Technique to Detect Chromogenic Signals During RNA In Situ Hybridization in Intact Tissue Specimens

Source: Outh-Gauer, S. et al. <a href="https://www.jove.com/video/59422" target="_blank">Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis.</a> J. Vis. Exp. (2019)
This video presents the protocol for signal detection during RNA chromogenic in situ hybridization. This technique can be used to diagnose an active oncogenic infection visually in an intact tissue specimen.

Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis

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