Dual ISH-IHC Application Protocol Using RNAScope®

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<div class="highlighted"> <aside class="container section clearfix" role="complementary"> <div> </div> </aside> </div> <div id="main" class="container-fluid container-wide "> <div class="row"> <div class="col-md-12"> <div> <div id="block-bio-techne-global-breadcrumbs" class="system_breadcrumb_block"> <nav role="navigation" aria-labelledby="system-breadcrumb"> <h2 id="system-breadcrumb" class="visually-hidden">Breadcrumb</h2> <ol> <li> <a href="/">Home</a> </li> <li> <a href="/resources">Resources</a> </li> <li> <a href="/resources/protocols-troubleshooting">All Protocols</a> </li> <li> ISH-IHC Protocol For Chromogenic Detection On Formalin Fixed Paraffin Embedded (FFPE) Tissue </li> </ol> </nav> </div> </div> </div> </div> <div class="row"> <div class="col-md-12"> <main> <section class="section"> <div> <div id="block-bio-techne-global-content" class="system_main_block"> <article> <h1><span>ISH-IHC Protocol for Chromogenic Detection on Formalin Fixed Paraffin Embedded (FFPE) Tissue</span> </h1> <div> <div class="bg-white border rounded protocol mb-5 p-4"> <div class="alert alert-warning"> This protocol is intended as a guide only, for full experimental details please read the reference provided. </div> <div class="row"> <aside class="col-md-3 text-md-right order-md-12"> <a href="https://resources.bio-techne.com/bio-techne-assets/docs/pdfs/FL-ish-ihc-protocol-NBv3.pdf" class="font-weight-bold" target="_blank"> <span class="icon-pdf-icon-flat text-error-red"></span> Open PDF </a> </aside> <div class="col-md-9"> <h2> </h2> </div> </div> <div> <div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h2>Materials for Chromogenic Detection on FFPE Tissue</h2></div> </div> </div> <div> <div class="paragraph paragraph--type--columns paragraph--view-mode--default"> <div class="list-grid list-grid-2"> <div class="row"> <div class="col-6 column-group"><h3>ISH Reagents from ACD</h3> <p>RNAscope<sup>®</sup> <a href="https://acdbio.com/store/rnascoper-2-5-hd-reagent-kit-red.html?utm_source=novusbio.com&utm_medium=referral&utm_campaign=sister-link&utm_content=ish-ihc-protocol-chromogenic-detection-ffpe-tissue" target="_blank">2.5 HD Reagent Kit-RED</a> (catalog # 322350) which includes:</p> <p><a href="https://acdbio.com/store/rnascoper-2-5-hd-detection-reagents-red.html?utm_source=novusbio.com&utm_medium=referral&utm_campaign=sister-link&utm_content=ish-ihc-protocol-chromogenic-detection-ffpe-tissue" target="_blank">HD Detection Kit (RED)</a> (catalog # 322360)</p> <p><a href="https://acdbio.com/store/rnascoper-wash-buffer-reagents.html?utm_source=novusbio.com&utm_medium=referral&utm_campaign=sister-link&utm_content=ish-ihc-protocol-chromogenic-detection-ffpe-tissue" target="_blank">Wash Buffer</a> (catalog # 310091)</p> <p><a href="https://acdbio.com/store/rnascoper-target-retrieval-reagents.html?utm_source=novusbio.com&utm_medium=referral&utm_campaign=sister-link&utm_content=ish-ihc-protocol-chromogenic-detection-ffpe-tissue" target="_blank">Target Retrieval Reagent</a> (catalog # 322000)</p> <p><a href="https://acdbio.com/store/rnascoper-h202-protease-plus-reagents.html?utm_source=novusbio.com&utm_medium=referral&utm_campaign=sister-link&utm_content=ish-ihc-protocol-chromogenic-detection-ffpe-tissue" target="_blank">Protease and Peroxidase Blocking Reagent</a> (catalog # 322330)</p></div> <div class="col-6 column-group"><h3>IHC Reagents</h3> <p>Normal Serum Blocking reagent</p> <p>Green HRP Chromogen</p> <p><a href="/search?search_type=products&keywords=IHC+Validated+Primary+Antibody">IHC Validated Primary Antibody</a></p> <p><a href="/search?search_type=products&keywords=HRP-+Polymer+Conjugated+Secondary+Antibody">HRP- Polymer Conjugated Secondary Antibody</a></p> <p>Gill’s Hematoxylin I (50%)</p> <p>VectaMount™ Mounting Medium (or equivalent)</p></div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h2>ISH Staining with RNAscope Assay</h2> <p>See RNAscope 2.5 HD Reagent Kit-RED Assay User Manual. The <a href="https://acdbio.com/sites/default/files/UM%20322452%20FFPE%20SamplePrep%20Rev%2003152017.pdf?utm_source=novusbio.com&utm_medium=referral&utm_campaign=sister-link&utm_content=ish-ihc-protocol-chromogenic-detection-ffpe-tissue" target="_blank">pretreatment conditions for FFPE tissues</a> outlined below for Target Retrieval step are only recommendations. It may be necessary to optimize protease digestion for the IHC target of choice. <em>Always follow user manual protocols that correspond to their appropriate kits purchased from Bio-Techne.</em></p></div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h2>Examples of Tissue and Target-Specific Pretreatment Condition Recommendations</h2> <figure class="responsive-figure-table"><table class="table table-striped"> <thead> <tr> <th class="bt-blue-table-header">Target</th> <th class="bt-blue-table-header">Antigen Retrieval</th> <th class="bt-blue-table-header">Protease Treatment</th> </tr> </thead> <tbody> <tr> <td><a href="/search?keywords=CD68&category=Primary%20Antibodies&applications=Dual%20RNAscope%20ISH-IHC">CD68</a></td> <td>15 min at 99°C in Oster® Steamer</td> <td>15 min at RT</td> </tr> <tr> <td><a href="/search?keywords=FoxP3&applications=Dual%20RNAscope%20ISH-IHC">FoxP3</a></td> <td>15 min at 99°C in Oster® Steamer</td> <td>15 min at RT</td> </tr> <tr> <td><a href="/search?keywords=CD8A&applications=Dual%20RNAscope%20ISH-IHC">CD8</a></td> <td>15 min at 99°C in Oster® Steamer</td> <td>15 min at RT</td> </tr> <tr> <td><a href="/search?keywords=Gata3&applications=Dual%20RNAscope%20ISH-IHC">Gata3</a></td> <td>15 min at 99°C in Oster® Steamer</td> <td>15 min at RT</td> </tr> <tr> <td><a href="/search?keywords=Vimentin&applications=Dual%20RNAscope%20ISH-IHC">Vimentin</a></td> <td>15 min at 99°C in Oster® Steamer</td> <td>20 min at RT</td> </tr> <tr> <td><a href="/search?keywords=CD52&applications=Dual%20RNAscope%20ISH-IHC">CD52</a></td> <td>15 min at 99°C in Oster® Steamer</td> <td>20 min at RT</td> </tr> </tbody> </table></figure> <p><em>*These recommended incubation times are for FFPE tonsil tissue. For less dense tissue (i.e. breast, normal lung, colon), decrease protease incubation time. For denser tissue (i.e. liver, muscle, or highly expressed protein targets (Vimentin)) longer protease incubation time is required.</em></p></div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h2>IHC Staining</h2> <p><em>IHC staining should be performed immediately following ISH chromogenic detection.</em></p> <ol> <li> <ul> <li>Note: PBS buffer decolorizes green chromogen. When using green chromogen, use only wash buffer provided by ACD for all washing steps. All steps are done at room temperature unless specified differently.</li> </ul> </li> <br> <li>Wash slides twice for 2 minutes in wash buffer.</li> <li>Incubate tissue sections for 15 minutes in 200 µL of Normal Serum Blocking Reagent. Tap solution off tissue sections - do not rinse slides.</li> <li>Using experimentally predetermined optimal conditions, incubate slides in 200 µL of primary antibody solution.</li> <li>Wash slides twice for 2 minutes in wash buffer.</li> <li>Apply 200 µL of HRP polymer-conjugated secondary antibody and incubate for 30 minutes.</li> <li>Wash slides three times for 2 minutes each in wash buffer.</li> <li>While your slides are washing, make a 1:50 working solution of a chromogen by mixing Green B (1 µL) with Green A components (49 µL). Make enough for 200 µL per slide.</li> <li>Apply 200 µL of green HRP solution to slides and incubate for 10-15 minutes.</li> <li>Quickly tap green HRP solution off slides and <em>briefly</em> rinse with distilled water. <ul> <li><strong>Note</strong>: Green substrate can lose its color by extended exposure to water.</li> </ul> </li> <li>Counterstain with 50% Hematoxylin I for 30 seconds. Gently rinse slides <em>briefly</em> with distilled water and place on bench top to dry or use oven to speed up drying procedure.</li> <li>Once dry, use a small amount of media to mount tissues.</li> </ol></div> </div> </div> <div> <div class="paragraph paragraph--type--_-2-media-to-text mb-4 paragraph--view-mode--default"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--50-50"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/protocols/ish-ihc-protocol-for-chromogenic-detection-on-ffpe-tissue-fig-1.jpeg" alt="Tonsil tissue probed for use in dual RNAscope ISH-IHC application displaying both RNA and protein expression of CD31/PECAM1." loading="lazy" > </div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_media_to_text:field_lg_sngl_frmtd_text_global"> <div><p><em>Example of <a href="/reagents/rnascope-ish-technology">dual RNAscope ISH-IHC</a> probed tissue. Formalin-fixed paraffin-embedded tissue sections of human metastatic tonsil were probed for CD31 mRNA (ACD RNAScope probe, catalog # 487381; <a href="https://acdbio.com/store/rnascoper-2-5-hd-reagent-kit-red.html?utm_source=novusbio.com&utm_medium=referral&utm_campaign=sister-link&utm_content=ish-ihc-protocol-chromogenic-detection-ffpe-tissue" target="_blank">Fast Red chromogen</a>, ACD catalog # 322500). Adjacent tissue section was processed for immunohistochemistry using <a href="/p/antibodies/cd31-pecam-1-antibody-jc-70a_nb600-562">mouse monoclonal CD31</a> (Catalog # NB600-562) at 1:25 dilution for 1 hour at room temperature followed by incubation with the anti-mouse <a href="/p/secondary-antibodies/mouse-igg-visucyte-hrp-polymer-antibody_vc001">IgG VisUCyte HRP Polymer Antibody</a> (Catalog # VC001) and DAB chromogen (yellow-brown). Tissue was counterstained with <a href="/p/staining-kits-reagents/hematoxylin_5222">Hematoxylin </a>(blue).</em></p></div> </div> </div> </div> </div> </div> </div> </div> </div> </div> <section> <div> <div id="accordion" class="paragraph mb-4 paragraph--type--references-container paragraph--view-mode--default"> </div> </div> </section> </div> </div> </article> </div> </div> </section> </main> </div> </div> </div> <div id="brandsbar-footer" class="bg-bt-primary"> <div class="container-wide container-fluid text-white"> <div class="row py-5"> <div class="footer-logo col-xl-3 col-lg-auto d-flex"> <img class="brand-bar-footer-logo" src="/themes/custom/bio_techne_global/logo.svg" alt="Bio-techne logo"> </div> <div class="col-xl-9 col-lg-auto"> <p class="ml-xl-4 bt-px_groteskbold footer-tagline">Global Developer, Manufacturer, and Supplier of High-Quality Reagents, Analytical Instruments, and Precision Diagnostics.</p> </div> <div class="included-brands col"> <span class="font-weight-bold 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