Immunohistochemistry (IHC): Products and Protocols for Success

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IHC serves as the foundational technique for spatial biology applications. Bio-Techne offers a complete line of high-quality IHC products, including antibodies from our R&D Systems and Novus Biologicals brands, and optimized IHC protocols that are key for generating accurate expression, localization, and distribution data.</p></div> </div> <div class="field_block:paragraph:h1_2_2_text_to_media:field_call_to_action_global"> <a href="/search?keywords=antibodies&applications=Immunohistochemistry" class="btn btn-primary" type="link"> Shop IHC Validated Antibodies </a> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> </div> </div> </div> </div> </div> </div> </div> </section> </div> </div> </header> </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="/applications">Applications</a> </li> <li> <a href="/applications/imaging">Imaging</a> </li> <li> Immunohistochemistry (IHC) </li> </ol> </nav> </div> </div> </div> </div> <main> <section class="section"> <div> <div id="block-bio-techne-global-content" class="system_main_block"> <article> <div> <div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3>On This Page</h3> <p><a href="#primary"><strong>Primary Antibodies</strong></a> |  <a href="#secondary"><strong>Secondaries & Detection Reagents</strong></a> |  <a href="#Tissue"><strong>Tissue Samples & Controls</strong></a> |  <a href="#support"><strong>Support Products</strong></a> |  <a href="#resources"><strong>Resources</strong></a></p></div> </div> </div> <div> <div class="paragraph paragraph--type--section paragraph--view-mode--default"> <div> <div> <div class="paragraph paragraph--type--card-paragraph paragraph--view-mode--default card-para-default"> <h2>Featured IHC Products</h2> <div class="row card_row"> <div class="col-md-4"> <div class="h-100"> <div class="bg-white border rounded flex-column d-flex h-100"> <a href="/applications/immunohistochemistry/spatial-tested-antibodies" > <div class="teaser-card-image-crop mb-3"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/applications/featured-multiplex-ab-fig-2-v3.jpg" alt="Detection of Cadherin in human colon cancer via Multiplex Immunofluorescence on COMET™" loading="lazy" > </div> </a> <a class="card_title_link" href="/applications/immunohistochemistry/spatial-tested-antibodies" > <h3 class="card_title_htag"> <div><h3>Multiplex IF Antibodies for Spatial Biology</h3></div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>Discover the combined power of R&D Systems<sup>TM</sup>, Novus Biologicals<sup>TM</sup> and Lunaphore brands for spatial biology with antibodies for Multiplex Immunofluorescence (mIF) on the high throughput, fully-automated COMET<sup>TM </sup>platform. </p></div> </div> <div class="card-bottom-link"> <div> <a href="/applications/immunohistochemistry/spatial-tested-antibodies" > View Primary Antibodies for Multiplex IF </a> </div> </div> </div> </div> </div> <div class="col-md-4"> <div class="h-100"> <div class="bg-white border rounded flex-column d-flex h-100"> <a href="/search?keywords=2BISH-IHC" > <div class="teaser-card-image-crop mb-3"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/mab7617-human-ki-67-mki67-mab-clone-1297a-dual-rnascope-ish-ihc-compatible-26102022142312.jpg" alt="Microscopic image of tissue sections of human breast cancer detecting Ki-67/MKI67 using Dual RNAscope ISH and IHC technique." loading="lazy" > </div> </a> <a class="card_title_link" href="/search?keywords=2BISH-IHC" > <h3 class="card_title_htag"> <div><h3>Antibodies for Spatial Multiomics</h3></div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>Amplify your multiomics research with Bio-Techne utilizing our Dual RNAscope ISH-IHC validated antibodies. By integrating staining strategies, simultaneous detection of RNA and protein within the same tissue section is possible.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/search?keywords=2BISH-IHC" > Explore Dual ISH-IHC Antibodies </a> </div> </div> </div> </div> </div> <div class="col-md-4"> <div class="h-100"> <div class="bg-white border rounded flex-column d-flex h-100"> <a href="/search?keywords=visucyte&sub_category=Cell%20and%20Tissue%20Staining%20Kits" > <div class="teaser-card-image-crop mb-3"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/antibodies-visucyte-hrp_card-update-v5.jpg" alt="antibodies-visucyte-hrp_card-update-v2.jpg" loading="lazy" > </div> </a> <a class="card_title_link" href="/search?keywords=visucyte&sub_category=Cell%20and%20Tissue%20Staining%20Kits" > <h3 class="card_title_htag"> <div><h3>IHC Staining Kits</h3></div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>Unlock research precision with Bio-Techne's VisUCyte™ HRP Polymer Detection Kits, delivering specific tissue staining at unprecedented speed. Rigorously tested for reproducibility by our in-house scientists, ensuring consistent results in every kit.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/search?keywords=visucyte&sub_category=Cell%20and%20Tissue%20Staining%20Kits" > Browse VisUCyte™ HRP Polymer Detection Kits </a> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> <div> <section class="paragraph paragraph--type--full-width-banner-with-image mb-4 paragraph--view-mode--default paragraph-background-color--"> <div class="banner_background_image" data-bgimage="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/immunohistochemistry-1110x350.jpg" style="background-repeat: no-repeat;"> <div> <div> <div> <div class="paragraph paragraph--type--_-2-text-to-media 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_text_to_media:field_lg_sngl_frmtd_text_global"> <div><h2>Discover Solutions To IHC Assays</h2> <p>In our New Complete IHC Guide</p> <p><a name="primary"></a></p></div> </div> <div class="field_block:paragraph:2_2_text_to_media:field_call_to_action_global"> <a href="/resources/literature/immunohistochemistry-ihc-handbook" class="btn btn-primary" type="link"> Download Handbook </a> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> </div> </div> </div> </div> </div> </div> </div> </div> </div> </section> </div> <div> <div class="paragraph paragraph--type--section paragraph--view-mode--default mb-4"> <div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h2>Immunohistochemistry Primary Antibodies</h2> <p>Selecting the right primary antibody is crucial for successful IHC experiments. Consider factors like species reactivity, tissue, subcellular localization, and post translational modifications (PTMs) when selecting a high-quality antibody that specifically binds the target antigen. Bio-Techne offers a robust catalog of highly sensitive antibodies, ensuring target specificity and minimal cross-reactivity. Explore our extensive data image library showcasing the performance of our IHC-validated antibodies across different tissues, applications, and species.</p></div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3>IHC Primary Antibodies by Species Reactivity</h3> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td><a href="/search?keywords=antibodies&category_attr=Primary%20Antibodies&applications=Immunohistochemistry">All Primary Antibodies</a></td> <td><a href="/search?keywords=antibodies&applications=Immunohistochemistry&category_attr=Primary%20Antibodies&species=Mouse">Mouse Primary Antibodies</a></td> </tr> <tr> <td><a href="/search?keywords=antibodies&applications=Immunohistochemistry&category_attr=Primary%20Antibodies&species=Human">Human Primary Antibodies</a></td> <td><a href="/search?keywords=antibodies&applications=Immunohistochemistry&category_attr=Primary%20Antibodies&species=Rat">Rat Primary Antibodies</a></td> </tr> </tbody> </table></figure> <p> </p></div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3><a name="secondary"></a>IHC Staining Protocols</h3> <p>IHC can be broadly classified into two forms based on the type of tissue processing involved: IHC- formalin-fixed, paraffin-embedded (FFPE), and IHC-frozen (Fr). Additionally, IHC results can be visualized by either immunofluorescence (IF) or by a chromogenic reaction. The tissue processing type and the intended detection method will inform the IHC staining workflow. Find protocols for each type of tissue and detection method below to expedite your IHC experiments.</p> <ul> <li><a href="/resources/protocols/multicolor-immunofluorescence-immunocytochemistry" target="_blank">Chromogenic Staining of FFPE Tissue Protocol</a></li> <li><a href="/resources/protocols-troubleshooting/chromogenic-ihc-staining-of-frozen-tissue-protocol" target="_blank">Chromogenic Staining of Frozen Tissue Protocol</a></li> <li><a href="/resources/protocols-troubleshooting/fluorescent-ihc-staining-of-ffpe-tissue" target="_blank">Fluorescent Staining of FFPE Tissue Protocol</a></li> <li><a href="/resources/protocols-troubleshooting/immunohistochemistry-frozen" target="_blank">Fluorescent Staining of Frozen Tissue Protocol</a></li> <li><a href="/resources/protocols-troubleshooting/fluorescent-ihc-staining-of-ffpe-tissue" target="_blank">Immunofluorescent IHC Staining of FFPE Tissue Protocol</a></li> </ul> <p> </p></div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--section paragraph--view-mode--default mb-4"> <div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h2>Secondary Antibodies and Detection Reagents</h2> <p>Secondary antibodies allow signal amplification and enhance sensitivity in IHC experiments by recognizing and binding to the primary antibodies. Both primary and secondary antibodies are conjugated to detection reagents, such as enzymes or fluorochromes to visualize antigen localization and expression.</p> <p> </p></div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3>IHC Secondary Antibodies by Detection Method</h3> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <thead> <tr> <th>Chromogenic Detection</th> <th>Fluorescent Detection</th> <th>Signal Amplification</th> </tr> </thead> <tbody> <tr> <td><a href="/search?keywords=primary%20antibodies&conjugate_form=HRP">HRP Primary Antibodies</a></td> <td><a href="/search?keywords=conjugated%20antibodies&category_attr=Primary%20Antibodies" target="_blank">Conjugated Primary Antibodies</a></td> <td><a href="/search?keywords=secondary%20antibodies&conjugate_form=Biotin">Biotin Conjugated Secondary Antibodies</a></td> </tr> <tr> <td><a href="/search?keywords=secondary%20antibodies&conjugate_form=HRP">HRP Secondary Antibodies</a></td> <td><a href="/search?keywords=conjugated%20antibodies&category_attr=Secondary%20Antibodies" target="_blank">Conjugated Secondary Antibodies</a></td> <td><a href="/search?keywords=HRP%20Polymer">HRP Polymer</a></td> </tr> </tbody> </table></figure> <p> </p></div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--card-paragraph paragraph--view-mode--default card-para-default"> <div class="row card_row"> <div class="col-md-6"> <div class="h-100"> <div class="bg-white border rounded flex-column d-flex h-100"> <a href="/search?keywords=tsa%20vivid" > <div class="teaser-card-image-crop mb-3"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/hela-cells-stain-with-dapi.jpg" alt="Hela Cells Stain with DAPI image" loading="lazy" > </div> </a> <a class="card_title_link" href="/search?keywords=tsa%20vivid" > <h3 class="card_title_htag"> <div><h2>TSA Vivid™ Fluorophore Kits</h2></div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>Enhance target detection with TSA Vivid dyes, known for their exceptional brightness, especially for low-abundance targets. These dyes significantly improve signal-to-noise ratios in spatial biology applications such as IHC, ICC and ISH. Optimized specifically for the <a href="/search?search_type=products&keywords=RNAscope™+Multiplex+Fluorescent+v2+Assay" target="_blank">RNAscope™ Multiplex Fluorescent v2 Assay</a>, TSA Vivid dyes ensure reliable and precise results.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/search?keywords=tsa%20vivid" > Browse TSA Vivid™ Fluorophore Kits </a> </div> </div> </div> </div> </div> <div class="col-md-6"> <div class="h-100"> <div class="bg-white border rounded flex-column d-flex h-100"> <a href="/search?keywords=primary%20antibody&brand=rnd&applications=Immunohistochemistry" > <div class="teaser-card-image-crop mb-3"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/ihc-ish-cd11b-expression.jpg" alt="Dual RNAscope ISH/IHC detection of CD11b expression in FFPE tissue sections of human lymph node " loading="lazy" > </div> </a> <a class="card_title_link" href="/search?keywords=primary%20antibody&brand=rnd&applications=Immunohistochemistry" > <h3 class="card_title_htag"> <div><h2>BSA and Azide Free Antibodies</h2></div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>Enhance your spatial phenotyping experiments with our comprehensive catalog of spatial-ready primary antibodies. Our IHC-validated antibodies are conjugation-ready (BSA and Azide-free) for convenient panel development in multiplexed spatial biology applications.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/search?keywords=primary%20antibody&brand=rnd&applications=Immunohistochemistry" > Browse BSA/Azide-Free IHC Antibodies </a> </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h2>Tissue Samples and Controls</h2> <p>Proper controls are critical to ensuring accurate interpretation of your IHC data. Bio-Techne offers tissue control slides that are important for identifying the source of any staining issues. Other controls like blocking peptides and isotype controls help verify the specificity of antigen-antibody interactions.</p> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td><a href="/search?&keywords=peptides%20&%20proteins&applications=Antibody%20Competition">Blocking Peptides</a></td> <td><a href="/search?keywords=isotype%20control&category_attr=Isotype%20Controls">Isotype Controls</a></td> <td><a href="/applications/imaging/immunohistochemistry/control-slides">Tissue Control Slides</a></td> </tr> </tbody> </table></figure> <p><em><a name="support"></a></em></p> <p> </p></div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h2>Support Products for IHC</h2><p>Bio-Techne offers a variety of support products to optimize your IHC workflows. Our products offer a wide array of benefits like reducing non-specific binding and background noise, vibrant staining, and high-throughput analysis. Explore all support products to enhance specificity, sensitivity, and overall experimental success in IHC.</p><figure class="responsive-figure-table"><table class="table table-bordered overflow-auto"><tbody><tr><td class="w-33"><a href="/search?&keywords=citrate+buffer">Antigen Retrieval Buffer</a></td><td><a href="/search?&keywords=serum&applications=Immunohistochemistry">Blocking Serum</a></td><td class="w-33"><a href="/p/supplemental/bovine-serum-albumin_5217">BSA</a></td></tr><tr><td><a href="/search?search_type=products&keywords=DAPI">DAPI</a></td><td><a href="/search?keywords=fluorescent%20dyes&sub_category=Janelia%20Fluor®%20Dyes">Janelia Fluor® Dyes</a></td><td><a href="/reagents/antibodies/conjugated-primary-antibodies/lightning-link-antibody-labeling-kits">Lightning-Link Antibody Labeling Kits</a></td></tr><tr><td><a href="/search?keywords=mitobrilliant">MitoBrilliant™ Dyes</a></td><td><a href="/search?&keywords=mounting+products">Mounting Media</a></td><td><a href="/applications/imaging/immunohistochemistry/tissue-microarrays">Tissue Microarrays (TMAs)</a></td></tr><tr><td><a href="https://www.bio-techne.com/search?keywords=tyramide%20signaling%20amplification">Tyramide Signaling Amplification (TSA</a>)</td><td> </td><td> </td></tr></tbody></table></figure><p><a name="resources"></a></p><p> </p></div> </div> </div> <div> <div class="paragraph paragraph--type--_-3-media-to-text paragraph--view-mode--default mb-4 p-3"> <div class="container-fluid container-wide"> <div class="layout layout--twocol-section layout--twocol-section--25-75"> <div class="layout__region layout__region--first"> <div class="hero-content-wrap"> <div class="field_block:paragraph:1_3_media_to_text:field_indv_media"> <div> <a href="https://www.bio-techne.com/resources/literature/fluorescent-dyes-and-probes-product-listing" title="Download Brochure" > <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/fluorescent_dyes_brochure_thumbnail.png" alt="Fluorescent Dyes and Probes Product Guide" loading="lazy" > </a> </div> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:1_3_media_to_text:field_header_2_global"> <div><h3>Fluorescent Dyes and Probes Brochure</h3></div> </div> <div class="field_block:paragraph:1_3_media_to_text:field_lg_sngl_frmtd_text_global"> <div><p>Fluorescence imaging incorporates many types of techniques for visualization of cells and sub-cellular components in live and fixed cells. Techniques include flow cytometry, IHC, ICC, FISH and many more. Our brochure can help you to select the most appropriate dye or probe to obtain the best results for your experiment, providing a background to the use of fluorescent dyes, probes and stains and listing our extensive product range.  </p></div> </div> <div class="field_block:paragraph:1_3_media_to_text:field_call_to_action_global"> <a href="/resources/literature/fluorescent-dyes-and-probes-product-listing" class="btn btn-primary" type="link"> Download Brochure </a> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--section paragraph--view-mode--default mb-4"> <div> <div> <div class="paragraph paragraph--type--header"> <div> <div><h2>Background Information, Understanding IHC, FAQs</h2></div> </div> </div> </div> <div> <div class="paragraph accordion mb-4 paragraph--type--extendable-paragraph paragraph--view-mode--default"> <div class="card-header rounded-top bg-white"> <a class="collapsed" data-toggle="collapse" href="#expPara-about-immunohistochemistry23" role="button" aria-expanded="false" aria-controls="expPara"> <h5>About Immunohistochemistry</h5> </a> </div> <div class="collapse" id="expPara-about-immunohistochemistry23" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <br/> <div><p>IHC is an antibody-based technique used to detect antigens in fixed tissue sections and provides semi-quantitative data about target protein expression, distribution, and localization. Briefly, samples are fixed to preserve cellular integrity and incubated with blocking reagents to prevent non-specific binding of the antibodies. Samples are then stained with primary, then secondary antibodies, and the signal is visualized with a microscope.</p> <p>The most challenging aspect of IHC is optimizing the experimental conditions required to generate a strong and specific signal for each antigen. Bio-Techne offers a variety of resources to streamline and troubleshoot your experiment to achieve the best results.</p></div> </div> </div> </div> </div> <div> <div class="paragraph accordion mb-4 paragraph--type--extendable-paragraph paragraph--view-mode--default"> <div class="card-header rounded-top bg-white"> <a class="collapsed" data-toggle="collapse" href="#expPara-understanding-ihc19" role="button" aria-expanded="false" aria-controls="expPara"> <h5>Understanding IHC</h5> </a> </div> <div class="collapse" id="expPara-understanding-ihc19" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <br/> <div><p><strong>Sample Preparation and Fixation Resources</strong></p> <p>In IHC experiments, fixation is essential for preserving tissue morphology and maintaining the antigenicity of target molecules. Most common fixatives for preserving protein targets in tissues include aldehydes, alcohols and acetone. Tissue preservation can be achieved through paraffin embedding or cryopreservation (freezing). Often the preservation method is closely associated with the type of fixation. Formalin-fixed tissues are commonly paraffin-embedded following fixation, while frozen tissue sections are fixed with alcohol following cryopreservation.</p> <p>Read more about these preparation methods and specific fixation techniques to effectively analyze tissue samples using IHC.</p> <ul> <li><a href="/applications/imaging/immunohistochemistry/sample-preparation-for-ihc-experiments">IHC Sample Preparation (Frozen vs. Paraffin)</a></li> <li><a href="/applications/imaging/immunohistochemistry/sample-fixation-for-ihc-icc">Sample Fixation for IHC</a></li> </ul> <p><strong>Antigen (Epitope) Retrieval Protocols and Resources</strong></p> <p>While fixation is important for preserving tissue morphology, it can adversely affect the detection of epitopes in immunohistochemistry (IHC) experiments. For example, formalin fixation can lead to the masking of the epitope of interest and inhibit the binding of the primary antibody. Antigen retrieval encompasses a range of techniques employed to reverse epitope masking and restore antigen-antibody binding.</p> <p>Learn effective antigen retrieval techniques:</p> <ul> <li><a href="/resources/protocols-troubleshooting/antigen-retrieval-hier">Heat-Induced Epitope Retrieval (HIER) Protocol</a></li> <li><a href="/resources/protocols-troubleshooting/antigen-retrieval-protocol-pier">Protease-induced Epitope Retrieval (PIER) Protocol</a></li> <li><a href="/resources/protocols-troubleshooting/antigen-retrieval-protocol-frozen-sections">Antigen Retrieval (Frozen Sections) Protocol</a></li> <li><a href="/applications/imaging/immunohistochemistry/epitope-retrieval">Epitope Retrieval (HIER vs. PIER)</a></li> </ul> <p><strong>Blocking Non-Specific Binding</strong></p> <p>Overcome the challenge of non-specific interactions in IHC experiments by using a blocking reagent like normal serum or protein solutions, such as BSA.</p> <ul> <li>Know more about <a href="/applications/imaging/immunohistochemistry/blocking-non-specific-binding">Blocking Non-Specific Binding in IHC</a></li> </ul> <p><strong>Antibody Selection and Detection Methods</strong></p> <p>Optimize primary antibody selection by considering protein characteristics, function, tissue, subcellular localization, and PTMs.</p> <ul> <li>Read tips on <a href="/applications/imaging/immunohistochemistry/primary-antibody-selection-optimization">IHC Primary Antibody Selection & Optimization</a></li> </ul> <p>Detection Methods: Antigen expression can be visualized in IHC using direct or indirect methods, generating chromogenic or fluorescent signals. Direct detection entails labeled primary antibodies, while indirect detection involves secondary antibodies. Both methods can be visualized through immunofluorescence (IF) or chromogenic reactions. For low-abundance antigens, signal amplification methods like biotin-based, HRP-polymer, or tyramide signal amplification (TSA) can be considered. Learn more about:</p> <ul> <li><a href="/applications/imaging/immunohistochemistry/ihc-detection" target="_blank">Detection Methods for IHC</a></li> <li><a href="/applications/imaging/immunohistochemistry/nuclear-cytosolic-staining">Cytosolic and Nuclear Staining </a></li> </ul> <p><strong>Read Tips and Techniques for accurate results.</strong></p> <ul> <li><a href="/applications/imaging/immunohistochemistry/ihc-paraffin-troubleshooting">IHC Troubleshooting Guide (FFPE)</a></li> <li><a href="/applications/imaging/immunohistochemistry/ihc-frozen-staining-trouble-shooting">IHC Troubleshooting Guide (Frozen)</a></li> </ul> <p><strong>Blocking Non-Specific Binding</strong></p> <p> </p></div> </div> </div> </div> </div> <div> <div class="paragraph accordion mb-4 paragraph--type--extendable-paragraph paragraph--view-mode--default"> <div class="card-header rounded-top bg-white"> <a class="collapsed" data-toggle="collapse" href="#expPara-faqs20" role="button" aria-expanded="false" aria-controls="expPara"> <h5>FAQs</h5> </a> </div> <div class="collapse" id="expPara-faqs20" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <br/> <div><p><strong>What is the difference between IHC and ICC?</strong></p> <p>IHC is the acronym for immuno<em>histo</em>chemistry and ICC is for immuno<em>cyto</em>chemistry. IHC always refers to using 2-15 µm thick tissue sections cut either frozen or paraffin-embedded and mounted on histological slides. In addition, IHC may refer to staining 150 – 300 µm thick tissue sections incubated free-floating and only after that mounted onto histological slides for microscopy examination. ICC defines the technique of staining cells either in suspension or adhered to a matrix or cytosmears rather than tissue sections. IHC and ICC can utilize similar immunofluorescence and enzymatic staining protocols. </p> <p><strong>What are the recommended working dilutions and volumes of primary antibodies?</strong></p> <p>There are different origins of primary antibodies developed for IHC applications including polyclonal, monoclonal, and recombinant antibodies. For most polyclonal antibodies, the working dilution range is from 1 to 15 µg/mL whereas for monoclonal and recombinant this range can be from 1 to 25 µg/mL. Polyclonal antibodies are used in lower concentrations to avoid non-specific background staining due to clones cross-reacting at higher concentrations with irrelevant tissue targets. Monoclonal and recombinant antibodies may be used at higher concentrations without the risk of producing background staining due to the lack of “contaminating” clones.</p> <p>It is of critical importance to prepare antibody working solutions sufficient for staining tissue sections selected for the IHC experiment:</p> <ul> <li>Preparing more than needed will be a waste of primary antibodies and making less than needed may result in not covering entire tissue sections with antibody solution for incubation.</li> <li>Depending on the area of the tissue section it may be required from 15 to 200 µL of antibody solution per slide with attached tissue section.</li> <li>The tissue section should be completely covered with antibody solution to avoid the formation of dry spots resulting in inconsistent tissue staining.</li> <li>Adding an excessive amount of antibody solution to the tissue sections can cause leakage from the slide and result in weak or staining and even lack of staining in parts of the tissue section tissue staining.</li> </ul> <p>To remove the guesswork from antibody solution calculations, it is usually recommended to use a calculating app (<a href="http://www.stainwell.com" target="_blank">www.stainwell.com</a>).</p> <p><strong>Which IHC controls are recommended? </strong></p> <p>IHC controls are always standard practice to make sure the staining signal is a result of the interaction of antibodies with its specific protein target rather than an artifact. Several types of controls can be used to address different issues. Before starting the IHC protocol it is always recommended to examine tissue sections under the microscope to assess the extent of tissue autofluorescence (if using immunofluorescence detection) and pigmentation (if using enzymatic chromogenic detection) which can obscure specific staining. For example, “aging pigment” lipofuscin, a product of oxidation of fatty acids, can be detected as yellow-brown speckles scattered throughout the cytoplasm resembling HRP converted DAB chromogen. Lipofuscin also has a very strong autofluorescence within a wide range (360–647 nm), emitting orange, green, yellow, and red light overlapping in color with many different fluorophores used for tissue detection. In addition, tissues may naturally autofluorescence which is caused by the presence of porphyrins and flavins.</p> <ol> <li><strong>No-primary antibody control: </strong>To rule out non-specific staining caused by the adsorption of secondary antibodies and detection reagents to tissue sections.</li> <li><strong>Primary antibody controls: </strong>These controls assess the specificity of primary antibodies. <ul> <li><strong>Tissue controls: </strong>Positive control includes staining of tissues known to express the antigen of interest whereas negative control includes staining of tissues lacking the antigen. If there is staining in the positive and negative control tissue, it may be indicative of the primary antibodies cross-reacting with non-specific tissue targets.</li> <li><strong>Cell controls:</strong> Like tissue controls but utilize (a) negative and positive cells. (b) transfected cells, and (c) cell knockouts.</li> <li><strong>In Situ Hybridization (ISH) control:</strong> For the detection of mRNA coding the protein of interest. ISH can be combined with IHC for the co-detection of both mRNA and the protein to make sure both signals are present in the same cells.</li> <li><strong>Absorption controls: </strong>To confirm that antibody interacts with a specific amino acid sequence of the target protein. Primary antibodies are mixed with peptide immunogens and then this mixture is used for staining tissue sections. It is expected to see no staining because specific peptides bound to antibody binding sites block them from reacting with the target protein. The presence of a signal may be indicative that antibodies cross-react with irrelevant tissue targets.</li> <li><strong>Western Blot controls:</strong> To determine if antibodies are detecting the protein of correct molecular weight in lysates of the same type of tissues used for IHC staining. Seeing multiple bands of wrong size band may be indicative of the cross-reactivity of antibodies with irrelevant proteins.</li> <li><strong>Isotype controls:</strong> These controls are used to ensure staining is not a result of using the same antibody class. For example, if the primary antibody is IgG1, then using IgG of the same class 1 will serve as a control.</li> </ul> </li> </ol> <p>Learn more about the <a href="/applications/imaging/immunohistochemistry/icc-ihc-if">Difference Between ICC and IHC</a>.</p> <p><strong>What should I do if there is no specific antibody available for my protein target of interest?</strong></p> <p>Epitope tags are proteins, peptides or small molecules that can be fused to your protein of interest using gene editing techniques and function as universal epitopes.  <a href="/search?keywords=antibodies&category_attr=Primary%20Antibodies&species=Epitope%20Tag">Epitope tag antibodies</a> that recognize these epitope tags are available from Bio-Techne. Learn more about<a href="/research-areas/epitope-tags"> Epitope Tags</a>. </p> <p> </p></div> </div> </div> </div> </div> <div> <div id="accordion" class="paragraph mb-4 paragraph--type--references-container paragraph--view-mode--default"> <div class="card-header"> <a class="collapsed" data-toggle="collapse" data-target="#refPara-123041" role="button" aria-expanded="true" aria-controls="refPara-123041"> <h5 class="d-inline">References</h5> </a> </div> <div id="refPara-123041" class="paragraph mb-4 paragraph--type--references-container paragraph--view-mode--default collapse accordion" aria-controls="refPara-123041"> <ol class="py-3"> <li class="py-2"> <p class="paragraph paragraph--type--individual-reference paragraph--view-mode--default image-and-text-default"> <strong>Buchwalow IB, Böcker W </strong> (2010) <a href="https://www.ncbi.nlm.nih.gov/pubmed/" title="View Abstract" target="_blank" rel="noreferrer"> Immunohistochemistry: basics and methods.</a> Springer </p> </li> <li class="py-2"> <p class="paragraph paragraph--type--individual-reference paragraph--view-mode--default image-and-text-default"> <strong>Lin F, Prichard J (eds.) </strong> (2015) <a href="https://www.ncbi.nlm.nih.gov/pubmed/" title="View Abstract" target="_blank" rel="noreferrer"> Handbook of practical immunohistochemistry, frequently asked questions.</a> Springer </p> </li> <li class="py-2"> <p class="paragraph paragraph--type--individual-reference paragraph--view-mode--default image-and-text-default"> <strong>Kalyuzhny, AE (ed.)</strong> (2017) <a href="https://www.ncbi.nlm.nih.gov/pubmed/" title="View Abstract" target="_blank" rel="noreferrer"> Signal Transduction Immunohistochemistry: Methods and Protocols.</a> Humana Press <strong>1</strong>:(Methods in Molecular Biology, 1554).. </p> </li> <li class="py-2"> <p class="paragraph paragraph--type--individual-reference paragraph--view-mode--default image-and-text-default"> <strong>Kalyuzhny, AE (ed.)</strong> (2022) <a href="https://www.ncbi.nlm.nih.gov/pubmed/" title="View Abstract" target="_blank" rel="noreferrer"> Signal Transduction Immunohistochemistry: Methods and Protocols.</a> Humana Press <strong>3</strong>:(Methods in Molecular Biology, 2593).. </p> </li> <li class="py-2"> <p class="paragraph paragraph--type--individual-reference paragraph--view-mode--default image-and-text-default"> <strong>Kalyuzhny, AE </strong> (2016) <a href="https://www.ncbi.nlm.nih.gov/pubmed/" title="View Abstract" target="_blank" rel="noreferrer"> Immunohistochemistry: Essential Elements and Beyond.</a> Springer (Techniques in Life Science and Biomedicine for the Non-Expert).. </p> </li> <li class="py-2"> <p class="paragraph paragraph--type--individual-reference paragraph--view-mode--default image-and-text-default"> <strong>Burry, RW </strong> (2010) <a href="https://www.ncbi.nlm.nih.gov/pubmed/" title="View Abstract" target="_blank" rel="noreferrer"> Immunocytochemistry, a practical guide for biomedical research. </a> Humana Press </p> </li> <li class="py-2"> <p class="paragraph paragraph--type--individual-reference paragraph--view-mode--default image-and-text-default"> <strong>Oliver C, Jamur MC (eds.) </strong> (2010) <a href="https://www.ncbi.nlm.nih.gov/pubmed/" title="View Abstract" target="_blank" rel="noreferrer"> Immunocytochemical methods and protocols.</a> Humana Press </p> </li> <li class="py-2"> <p class="paragraph paragraph--type--individual-reference paragraph--view-mode--default image-and-text-default"> <strong>Mondal PP, Diaspro A </strong> (2014) <a href="https://www.ncbi.nlm.nih.gov/pubmed/" title="View Abstract" target="_blank" rel="noreferrer"> Fundamentals of fluorescence microscopy exploring life with light.</a> Springer, New York </p> </li> <li class="py-2"> <p class="paragraph paragraph--type--individual-reference paragraph--view-mode--default image-and-text-default"> <strong>Verveer PJ (ed.) </strong> (2015) <a href="https://www.ncbi.nlm.nih.gov/pubmed/" title="View Abstract" target="_blank" rel="noreferrer"> Advanced fluorescence microscopy, methods, and protocols.</a> Humana Press </p> </li> <li class="py-2"> <p class="paragraph paragraph--type--individual-reference paragraph--view-mode--default image-and-text-default"> <strong>Diaspro A (ed.) </strong> (2011) <a href="https://www.ncbi.nlm.nih.gov/pubmed/" title="View Abstract" target="_blank" rel="noreferrer"> Optical fluorescence microscopy, from the spectral to the nano dimension.</a> Springer </p> </li> <li class="py-2"> <p class="paragraph paragraph--type--individual-reference paragraph--view-mode--default image-and-text-default"> <strong>Chiarini-Garcia H, Melo R (eds.)</strong> (2011) <a href="https://www.ncbi.nlm.nih.gov/pubmed/" title="View Abstract" target="_blank" rel="noreferrer"> Light microscopy, methods, and protocols.</a> Humana Press </p> </li> <li class="py-2"> <p class="paragraph paragraph--type--individual-reference paragraph--view-mode--default image-and-text-default"> <strong>Wiley, Chichester</strong> (1993) <a href="https://www.ncbi.nlm.nih.gov/pubmed/" title="View Abstract" target="_blank" rel="noreferrer"> Immunohistochemistry II.</a> ACCuello (ed.) ISBN-13:9780471934608. </p> </li> <li class="py-2"> <p class="paragraph paragraph--type--individual-reference paragraph--view-mode--default image-and-text-default"> <strong>Johnstone AP, Turner MW (eds.) </strong> (1997) <a href="https://www.ncbi.nlm.nih.gov/pubmed/" title="View Abstract" target="_blank" rel="noreferrer"> Immunochemistry: a practical approach.</a> IRL Press, Oxford. <strong>2</strong>:ISBN:0199636079. </p> </li> </ol> </div> </div> </div> </div> </div> </div> </div> </div> </article> </div> </div> </section> </main> </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, 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Immunohistochemistry (IHC): Products and Protocols for Success