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<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:h1_2_2_text_to_media_multi_cta:field_indv_h1"> <div><h1>Immunocytochemistry (ICC)</h1></div> </div> <div class="field_block:paragraph:h1_2_2_text_to_media_multi_cta:field_lg_sngl_frmtd_text_global"> <div><p>Immunocytochemistry (ICC), often referred to as immunofluorescence (IF), is an immunostaining technique that detects antigens in cultured or primary cells via light microscopy. Fluorescent conjugated antibodies are used in multicolor ICC experiments.</p></div> </div> <div class="field_block:paragraph:h1_2_2_text_to_media_multi_cta:field_call_to_action_global"> <a href="/search?keywords=antibodies&applications=Immunocytochemistry/%20Immunofluorescence" class="btn btn-primary btn-cta mr-3 mb-3" type="link"> Find ICC 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> Immunocytochemistry (ICC) </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><p><a href="#primary">ICC Primary Antibodies</a> | <a href="#secondary">Secondary Antibodies and Detection Reagents</a> | <a href="#controls">Controls</a> | <a href="#support">Support Products</a> | <a href="#resources">ICC Protocols and Other Resources</a>| <a href="#background">Background Information</a></p></div> </div> </div> <div> <div class="paragraph paragraph--type--card-paragraph paragraph--view-mode--default card-para-default"> <h2>Featured ICC 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="/p/stem-cell/human-pluripotent-stem-cell-marker-antibody-panel-plus_sc009" > <div class="teaser-card-image-crop mb-3"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/human-pluripotent-stem-cell-marker-antibody-panel-plus-teaser.jpg" alt="Human iPSCs labeled with antibodies from the Human Pluripotent Stem Cell Marker Antibody Panel Plus" loading="lazy" > </div> </a> <a class="card_title_link" href="/p/stem-cell/human-pluripotent-stem-cell-marker-antibody-panel-plus_sc009" > <h3 class="card_title_htag"> <div><h3>Verify Stemness of iPSCs</h3></div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>Analyzing human stem pluripotency using marker antibodies allows the identification and expansion of high quality, undifferentiated stem cell populations. Provides increased confidence in pluripotent status through the use of multiple markers.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/p/stem-cell/human-pluripotent-stem-cell-marker-antibody-panel-plus_sc009" > View PSC Marker Antibody Panel </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=antibodies&category_attr=Primary%20Antibodies&applications=Immunocytochemistry/%20Immunofluorescence" > <div class="teaser-card-image-crop mb-3"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/caveolin-1-detection-by-icc.jpg" alt="Detection of caveolin-1 in human cervical epithelial carcinoma cell line by ICC" loading="lazy" > </div> </a> <a class="card_title_link" href="/search?keywords=antibodies&category_attr=Primary%20Antibodies&applications=Immunocytochemistry/%20Immunofluorescence" > <h3 class="card_title_htag"> <div><h3>Signal Transduction in Focus</h3></div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>Visualize intracellular signaling events with antibodies validated for ICC. Whether its synapse formation and subsequent signalling cascade or the watching the inner workings of the mitochondria, we have a range of antibodies for signal transduction.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/search?keywords=antibodies&category_attr=Primary%20Antibodies&applications=Immunocytochemistry/%20Immunofluorescence" > Find ICC/IF Antibodies for Intracellular Signalling </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="/p/staining-kits-reagents/tissue-clearing-pro-organoid_7390" > <div class="teaser-card-image-crop mb-3"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/tissue_clearing_pro_organoid_fluor.jpg" alt="Vibrant, fluorescent organoid utilizing tissue clearing to render the organoid transparent" loading="lazy" > </div> </a> <a class="card_title_link" href="/p/staining-kits-reagents/tissue-clearing-pro-organoid_7390" > <h3 class="card_title_htag"> <div><h3>Get Clearer Organoid Images</h3></div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>Visualize intracellular signalling events with antibodies validated for ICC. Whether its synapse formation and subsequent signalling cascade or the watching the inner workings of the mitochondria, we have a range of antibodies for signal transduction.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/p/staining-kits-reagents/tissue-clearing-pro-organoid_7390" > View Tissue Clearing-Pro Organoid Kit </a> </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="primary"></a></p></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>Immunocytochemistry (ICC) Primary Antibodies</h2></div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p>Successful ICC experiments start with specific and validated primary antibodies. Factors like species reactivity, clonality, and host species are critical considerations for <a href="/applications/imaging/immunocytochemistry/antibody-selection-for-icc-if"><strong>antibody selection</strong></a>. To maximize your ICC staining signal, it is important to ensure antibody specificity while minimizing cross-reactivity. Bio-Techne’s extensive data image library showcases the performance of our ICC/IF validated antibodies across different cell types and species.</p> <p>For ICC tips and guidance see our <a href="#resources"><strong>ICC Protocols</strong></a>.</p> <h3><strong>ICC Primary Antibodies by Species Reactivity</strong></h3> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td><a href="/search?keywords=antibodies&applications=Immunocytochemistry/%20Immunofluorescence&category_attr=Primary%20Antibodies">All Primary Antibodies</a></td> <td><a href="/search?keywords=antibodies&applications=Immunocytochemistry/%20Immunofluorescence&category_attr=Primary%20Antibodies&species=Mouse">Mouse Primary Antibodies</a></td> </tr> <tr> <td><a href="/search?keywords=antibodies&applications=Immunocytochemistry/%20Immunofluorescence&category_attr=Primary%20Antibodies&species=Human">Human Primary Antibodies</a> </td> <td><a href="https://www.bio-techne.com/search?keywords=antibodies&applications=Immunocytochemistry/%20Immunofluorescence&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--individual-button mb-4 paragraph--view-mode--default w-100"> <div class="row"> <div class="col"> <a href="/reagents/antibodies/epitope-tag-antibodies" class="btn btn-primary float-left" type="link" > Find Out About Our Epitope Tag Antibodies </a> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3>Antibodies for Multicolor ICC</h3> <p>Bio-Techne is here to meet your multicolor ICC needs. We offer an unparalleled catalog of <a href="/reagents/antibodies/conjugated-primary-antibodies"><strong>fluorescent conjugated antibodies</strong></a>,<strong> </strong>including primary antibodies conjugated to photostable dyes such as Alexa Fluor® 647 and Alexa Fluor® 488. </p> <p>Find pre-conjugated primary antibodies below: </p> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td><a href="/search?search_type=products&keywords=primary%20antibody&conjugate_form=Alexa%20Fluor%20488">Alexa Fluor® 488</a></td> <td><a href="/search?=products&keywords=primary%20antibody&rnd_applications=Immunocytochemistry/Immunofluorescence&conjugate_form=Janelia%20Fluor%20549">Janelia Fluor® 549</a></td> <td><a href="/search?keywords=primary%20antibody&rnd_applications=Immunocytochemistry/Immunofluorescence&conjugate_form=DyLight%20550">DyLight<sup>TM</sup> 550</a></td> </tr> <tr> <td><a href="/search?search_type=products&keywords=primary%20antibody&conjugate_form=Alexa%20Fluor%20647">Alexa Fluor® 647</a></td> <td><a href="/search?=products&keywords=primary%20antibody&rnd_applications=Immunocytochemistry/Immunofluorescence&conjugate_form=Janelia%20Fluor%20646">Janelia Fluor® 646</a></td> <td><a href="/search?search_type=products&keywords=primary%20antibody&conjugate_form=Biotin">Biotin</a></td> </tr> </tbody> </table></figure></div> </div> </div> <div> <div class="paragraph paragraph--type--individual-button mb-4 paragraph--view-mode--default w-100"> <div class="row"> <div class="col"> <a href="/reagents/fluorescent-dyes-probes/fluorescent-dyes/janelia-fluor" class="btn btn-primary float-left" type="link" > Learn More About Janelia Fluor® Dyes </a> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="secondary"></a></p></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>Secondary Antibodies and Detection Reagents</h2></div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p>ICC staining can be visualized using direct staining with a conjugated primary antibody or indirect detection using a secondary conjugated antibody. In indirect IF detection, a fluorochrome-labeled secondary antibody is required to detect the primary antibody-antigen complex. If you are using indirect detection methods, it is important to ensure the primary antibodies have different host species during primary antibody selection. Other considerations for secondary antibody selection include primary antibody subclass (eg, IgG, IgM, IgA) and targeting the Heavy Chain (Hc) only, Light Chain (Lc) only, or both Heavy and Light Chain (H+L).</p> <p>Indirect detection methods can improve detection of low abundance targets due to multiple secondary antibodies binding to one primary antibody. <a href="#background">Learn more about the differences between direct and indirect detection.</a></p> <h3>ICC Secondary Antibodies by Reactivity</h3> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td><a href="/search?keywords=antibodies&category_attr=Secondary%20Antibodies&species=Goat">Anti-Goat</a></td> <td><a href="/search?keywords=antibodies&category_attr=Secondary%20Antibodies&species=Mouse">Anti-Mouse</a></td> <td><a href="/search?keywords=antibodies&category_attr=Secondary%20Antibodies&species=Rat">Anti-Rat</a></td> </tr> <tr> <td><a href="/search?keywords=antibodies&category_attr=Secondary%20Antibodies&species=Human">Anti-Human</a></td> <td><a href="/search?keywords=antibodies&category_attr=Secondary%20Antibodies&species=Rabbit">Anti-Rabbit</a></td> <td><a href="/search?keywords=antibodies&category_attr=Secondary%20Antibodies&species=Sheep">Anti-Sheep</a></td> </tr> </tbody> </table></figure> <h3>Detection Reagents</h3> <p>In addition to antibody staining, <a href="/reagents/fluorescent-dyes-probes">fluorescent dyes and probes</a> can be used to further investigate cellular structures and visualize cellular morphology.</p> <figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <thead> <tr> <th class="bt-blue-table-header">Cellular Structure</th> <th class="bt-blue-table-header">Reagents</th> <th class="bt-blue-table-header">Use</th> </tr> </thead> <tbody> <tr> <td>Nucleus</td> <td><a href="/p/fluorescent-probes-dyes/dapi-solution_nbp2-31156">DAPI</a>, <a href="/p/fluorescent-probes-dyes/draq5-tm-_nbp2-81125">DRAQ5<sup>TM</sup></a></td> <td>Live or fixed cells; membrane permeable</td> </tr> <tr> <td>Nucleus</td> <td><a href="/p/fluorescent-probes-dyes/draq7-tm-_nbp2-81126">DRAQ7<sup>TM</sup></a></td> <td>Live or fixed cells; will not stain intact live cells, can be used for viability</td> </tr> <tr> <td>Cytoplasm</td> <td><a href="/p/fluorescent-probes-dyes/draq9-tm-_nbp2-81128">DRAQ9<sup>TM</sup></a></td> <td>Live or fixed cells; membrane permeable</td> </tr> <tr> <td>Mitochondria</td> <td><a href="https://www.bio-techne.com/p/fluorescent-probes-dyes/mitobrilliant-646_7700">MitoBrilliant<sup>TM</sup> 646</a></td> <td>Live or fixed cells</td> </tr> <tr> <td>Mitochondria</td> <td><a href="https://www.bio-techne.com/p/fluorescent-probes-dyes/mitobrilliant-live-646_7417">MitoBrilliant<sup>TM</sup> Live 646</a>, <a href="https://www.bio-techne.com/p/fluorescent-probes-dyes/mitobrilliant-live-549_7693">MitoBrilliant<sup>TM</sup> Live 549</a></td> <td>Live cells; activity dependent on membrane potential (Δψm)</td> </tr> </tbody> </table></figure> <p> </p></div> </div> </div> <div> <div class="paragraph paragraph--type--individual-button mb-4 paragraph--view-mode--default w-100"> <div class="row"> <div class="col"> <a href="/reagents/fluorescent-dyes-probes" class="btn btn-primary float-left" type="link" > Explore All Fluorescent Dyes and Probes </a> </div> </div> </div> </div> </div> </div> </div> <div> <div class="thin-banner banner_wrapper"> <div class="banner-dark"> <div> <div><h2>Improve the brightness and performance of your ICC</h2></div> <a href="/search?keywords=TSA%20Vivid%E2%84%A2" > <button type="button" class="btn btn-primary"> TSA Vivid Fluorophore Kits </button> </a> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="controls"></a></p></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>ICC Controls</h2></div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p>Proper experimental controls are critical to ensuring accurate interpretation of your ICC data. ICC controls include background staining controls and antibody specificity controls.</p> <h3>Antibody Specificity Controls</h3> <p>Bio-Techne performs rigorous testing on all ICC validated antibodies. Our extensive data image library showcases our dedicate to antibody quality.  Some common methods to verify antibody specificity are outlined below, including knockout (KO) cell line, positive and negative cell lines, protein stimulation, and RNA-protein codetection. Bio-Techne also offers controls such as <a href="/applications/imaging/immunocytochemistry/blocking-icc-if">blocking peptides</a>.</p></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-knockout-ko-cell-line-control4" role="button" aria-expanded="false" aria-controls="expPara"> <h5>Knockout (KO) Cell Line Control</h5> </a> </div> <div class="collapse" id="expPara-knockout-ko-cell-line-control4" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/icc-krt18-detection-in-hela-cells.jpg" alt="ICC detection of KRT18 in HeLa cells" loading="lazy" > </div> <br/> <div><p>Knockout ICC Control. Detection of KRT18 in wild-type HeLa cells (positive) control and in HeLa cells in which KRT18 was knocked out (negative control). Notice lack of staining in KO cells which is indicative for the specificity of primary antibodies. Cells were counterstained with <a href="/p/fluorescent-probes-dyes/dapi-solution_nbp2-31156">DAPI</a> (NBP2-31156).</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-positive-and-negative-cell-line-control1" role="button" aria-expanded="false" aria-controls="expPara"> <h5>Positive and Negative Cell Line Control</h5> </a> </div> <div class="collapse" id="expPara-positive-and-negative-cell-line-control1" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/icc-beta-iii-tubulin-detection.jpg" alt="Detection of neuron-specific beta-III tubulin in SH-SY5Y cells by ICC" loading="lazy" > </div> <br/> <div><p>Positive and Negative Cell Line Control. Detection of neuron-specific β-III Tubulin in SH-SY5Y cells known to express this protein. No staining was observed in HEL92.1.7 cells which do not express this protein. Cells were counterstained with <a href="/p/fluorescent-probes-dyes/dapi-solution_nbp2-31156">DAPI</a> (NBP2-31156).</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-stimulation-controls20" role="button" aria-expanded="false" aria-controls="expPara"> <h5>Stimulation Controls</h5> </a> </div> <div class="collapse" id="expPara-stimulation-controls20" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/icc-hif-1-alpha-detection.jpg" alt="Detection of HIF-1alpha in HELA cells by ICC" loading="lazy" > </div> <br/> <div><p>Protein Stimulation Control. HeLa cells underwent hypoxia treatment leading to upregulation of HIF-1α protein in cell nuclei (left), whereas no signal was detected in untreated cells. Cells were counterstained with <a href="/p/fluorescent-probes-dyes/dapi-solution_nbp2-31156">DAPI</a> (NBP2-31156).</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-mrna-and-protein-co-detection8" role="button" aria-expanded="false" aria-controls="expPara"> <h5>mRNA and Protein Co-detection</h5> </a> </div> <div class="collapse" id="expPara-mrna-and-protein-co-detection8" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/icc-and-rnascope-egfr-codetection.jpg" alt="Co-detection of EGFR protein and RNA by ICC and RNAscope" loading="lazy" > </div> <br/> <div><p>RNA-Protein Co-detection Control. Co-detection of epidermal growth factor receptor (EGFR) mRNA (red) using RNAscope<sup>TM</sup> protocol and EGFR protein (green) by ICC in the same cells. Co-localization of mRNA and its protein within the same cells confirms specificity of antibodies. </p></div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3>Background Staining Controls</h3> <p>Background staining can be a confounding factor when visualizing ICC images. If using an indirect detection method, it’s important to include a no primary antibody control. This can be accomplished by leaving one sample out of the primary antibody incubation step and then continuing to treat that sample with secondary antibody and any additional detection reagents.</p> <p><a href="/search?keywords=isotype%20control">Isotype controls</a> are used as negative controls to help differentiate non-specific background signal from specific antibody signal. Isotype controls are primary antibodies that lack specificity to the target, but match the class and type of the primary antibody used in the application.</p></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-isotype-controls25" role="button" aria-expanded="false" aria-controls="expPara"> <h5>Isotype Controls</h5> </a> </div> <div class="collapse" id="expPara-isotype-controls25" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/icc-afp-isotype-control.jpg" alt="ICC detection of AFP in HepG2 cells with non-immune IgG1 antibody isotype control" loading="lazy" > </div> <br/> <div><p>HepG2 cells were incubated either with <a href="/p/antibodies/human-mouse-alpha-fetoprotein-afp-antibody-189502_mab1368">mouse anti-human AFP antibody</a> (MAB1368) IgG1 isotype or with <a href="/p/isotype-controls/mouse-igg-1-isotype-control_mab002">non-immune IgG1 antibody</a> (MAB002) both at 25 μg/mL working dilution. Strong specific cell labeling in the cytoplasm can be seen with MAB1368 antibody, whereas there was no staining in cells incubates with IgG1 isotype control.</p></div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="support"></a></p></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>Support Products for ICC</h2></div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><figure class="responsive-figure-table"><table class="overflow-auto table table-bordered"> <tbody> <tr> <td><a href="/search?search_type=products&keywords=normal+serum">Normal Serum</a></td> <td class="w-33"><a href="/t/pap-pen">Pap Pen</a></td> <td><a href="/t/mounting-medium">Mounting Media</a></td> </tr> <tr> <td><a href="/p/supplemental/pbs-100-tablets_5564">PBS Buffer Tablets</a></td> <td><a href="/p/supplemental/bovine-serum-albumin_5217">BSA</a></td> <td> </td> </tr> </tbody> </table></figure></div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="resources"></a></p></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>Key Resources for ICC</h2></div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3> </h3> <h3>ICC Protocols and Application Support</h3> <p>Learn more about the basics of ICC and the differences between direct and indirect detection in our protocols and methods pages. </p> <p> </p></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"><h4>Protocols</h4> <ul> <li><a href="/resources/protocols-troubleshooting/immunocytochemistry">ICC Protocol</a></li> <li><a href="/resources/protocols-troubleshooting/icc">ICC Video Protocol</a></li> <li><a href="/resources/protocols-troubleshooting/multicolor-immunofluorescence-immunocytochemistry">Multicolor ICC/IF Protocols</a></li> <li><a href="/applications/imaging/immunocytochemistry/troubleshooting">ICC Troubleshooting Guide</a></li> <li><a href="https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-icc-staining-cells-coverslips">Protocol for the Preparation and Fluorescent ICC Staining of Cells on Coverslips</a></li> <li><a href="https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-icc-staining-non-adherent-cells">Protocol for the Preparation and Fluorescent ICC Staining of Non-adherent Cells</a></li> <li><a href="https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-icc-staining-stem-cells-coverslips">Protocol for the Preparation and Fluorescent ICC Staining of Stem Cells on Coverslips</a></li> </ul> <p> </p></div> <div class="col-6 column-group"><h4>Application Support</h4> <ul> <li><a href="/applications/imaging/immunocytochemistry/sample-preparation-for-icc-if-experiments">Sample Preparation for ICC/IF Experiments</a></li> <li><a href="/applications/immunocytochemistry/fixation-and-permeabilization-in-icc-if">Fixation and Permeabilization in Immunocytochemistry/Immunofluorescence (ICC/IF)</a></li> <li><a href="/applications/immunohistochemistry/sample-fixation-for-ihc-icc">IHC/ICC Sample Fixation (Formalin vs. Alcohol)</a></li> <li><a href="/applications/imaging/immunocytochemistry/blocking-icc-if">Blocking ICC/IF</a></li> <li><a href="/applications/immunohistochemistry/nuclear-cytosolic-staining">Cytosolic and Nuclear Staining</a></li> <li><a href="/applications/imaging/immunocytochemistry/counterstaining-and-mounting-in-icc-if">Counterstaining and Mounting in ICC/IF</a></li> <li><a href="/applications/imaging/immunocytochemistry/controls-for-icc-if-experiments">Controls for ICC/IF Experiments</a></li> <li><a href="/applications/imaging/immunocytochemistry/selection-of-fluorochromes-for-multicolor-icc-if">Selection of Fluorochromes for Multicolor ICC/IF</a></li> <li><a href="/applications/imaging/immunocytochemistry/troubleshooting">Immunocytochemistry Troubleshooting</a></li> </ul></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/banners/bt-hero-gradient-research-areas1.png" style="background-repeat: no-repeat;"> <div> <div> <div> <div class="paragraph paragraph--type--_-2-text-to-media 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View the excitation and emission spectra for our fluorescent dye range and other commonly used dyes.</p></div> </div> <div class="field_block:paragraph:2_2_text_to_media:field_call_to_action_global"> <a href="/resources/spectra-viewer" class="btn btn-primary" type="link"> View Excitation & Emission Spectra </a> </div> </div> </div> <div class="layout__region layout__region--second"> <div class="hero-content-wrap"> <div class="field_block:paragraph:2_2_text_to_media:field_indv_media"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/Spectra-Viewer-dark-1200.png" alt="Spectra Viewer" loading="lazy" > </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </div> </section> </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="d-flex flex-column h-100"> <a href="/resources/literature/fluorescent-dyes-and-probes-product-listing" > <div class="card-img-top text-center mb-3" style="background-repeat: no-repeat; background-color: ; height:200px; width: 100%;"> <div class="teaser-valign-container" style=""> <div class="valign"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/fluorescent_dyes_brochure_teaser_card.png" alt="Fluorescent Probes and Dyes Brochure from Tocris" loading="lazy" > </div> </div> </div> </a> <a class="card_title_link" href="/resources/literature/fluorescent-dyes-and-probes-product-listing" > <h3 class="card_title_htag"> <div><h3>Fluorescent Dyes and Probes Brochure</h3></div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>Providing a background to the use of fluorescent dyes, probes and stains and listing our extensive product range, our brochure can help you to select the most appropriate dye or probe to obtain the best results for your experiment.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/resources/literature/fluorescent-dyes-and-probes-product-listing" > Download Brochure </a> </div> </div> </div> </div> </div> <div class="col-md-6"> <div class="h-100"> <div class="d-flex flex-column h-100"> <a href="/resources/literature/immunocytochemistry-icc-handbook" > <div class="card-img-top text-center mb-3" style="background-repeat: no-repeat; background-color: ; height:200px; width: 100%;"> <div class="teaser-valign-container" style=""> <div class="valign"> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/icc-handbook-cover-img-v2.png" alt="ICC Handbook Cover" loading="lazy" > </div> </div> </div> </a> <a class="card_title_link" href="/resources/literature/immunocytochemistry-icc-handbook" > <h3 class="card_title_htag"> <div><h3>Immunocytochemistry (ICC) Handbook</h3></div> </h3> </a> <div class="card_description flex-grow-1"> <div><p>This Immunocytochemistry (ICC) Handbook offers an introduction to ICC focusing on multicolor ICC/IF, including detailed protocols and troubleshooting tips. It is a great resource for researchers new to ICC/IF as well as experienced users looking to brush up on their skills.</p></div> </div> <div class="card-bottom-link"> <div> <a href="/resources/literature/immunocytochemistry-icc-handbook" > Request Your Copy </a> </div> </div> </div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><h3>ICC Related Blog Posts </h3> <ul> <li><a href="/applications/imaging/immunocytochemistry/antibody-selection-for-icc-if">Antibody Selection for ICC/IF </a></li> <li><a href="/resources/blogs/five-key-tips-for-a-better-multicolor-immunofluorescence-staining">Five Key Tips for a Better Multicolor Immunofluorescence Staining</a></li> </ul></div> </div> </div> </div> </div> </div> <div> <div class="paragraph paragraph--type--freeform mb-4 paragraph--view-mode--default"> <div><p><a name="background"></a></p></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</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-what-is-immunocytochemistry-icc10" role="button" aria-expanded="false" aria-controls="expPara"> <h5>What is Immunocytochemistry (ICC)?</h5> </a> </div> <div class="collapse" id="expPara-what-is-immunocytochemistry-icc10" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/multicolor-icc-if-scheme.jpg" alt="Multicolor immunocytochemistry scheme for indirect protein detection " loading="lazy" > </div> <br/> <div><p>ICC is a widely used form of immunostaining, for visualizing specific proteins in cells, using labeled antibodies that selectively bind the target protein in situ. Visualization is via fluorescence microscopy and enables assessment of whether and where a cell expresses a particular protein. The ICC technique uses cells that are isolated from tissue, such as cells in culture, whereas <a href="/methods/immunohistochemistry">immunohistochemistry (IHC)</a> uses sections of tissue. Learn more about the <a href="/applications/imaging/immunohistochemistry/icc-ihc-if">difference between ICC and IHC</a>.</p> <p>The illustration above depicts a multicolor ICC/IF scheme for indirect protein detection. HeLa cells were probed with <a href="/p/antibodies/ki67-mki67-antibody-1297a_nbp2-54791">anti-Ki67</a> (nuclear protein; NBP2-54791) and <a href="/p/antibodies/alpha-tubulin-antibody-dm1a_nb100-690">anti-α-tubulin</a> (microtubular protein; NB100-690) followed by detection with DyLight 488 (green) and DyLight 550 (red) conjugated secondary antibodies, respectively. Fixed cells were counterstained with the nuclear dye, <a href="/p/fluorescent-probes-dyes/dapi-solution_nbp2-31156">DAPI</a> (blue; NBP2-31156).</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-direct-and-indirect-detection-methods7" role="button" aria-expanded="false" aria-controls="expPara"> <h5>Direct and Indirect Detection Methods</h5> </a> </div> <div class="collapse" id="expPara-direct-and-indirect-detection-methods7" aria-expanded="false" aria-controls="expPara"> <div class="card-body p-4"> <div> <img src="https://resources.bio-techne.com/bio-techne-assets/images/research-areas/direct-and-indirect-methods-800px.jpg" alt="Illustration of direct and indirect detection methods in ICC and IHC" loading="lazy" > </div> <br/> <div><p>In fluorescence-based immunodetection, antibodies are conjugated to fluorochromes which emit light upon excitation with light at a specific wavelength. While detection typically involves either a labeled primary or secondary antibody, using a biotinylated antibody followed by fluorochrome-labeled streptavidin improves assay sensitivity.</p> <p><strong>Direct and Indirect Detection</strong></p> <p>Direct detection in multicolor ICC/IF uses primary antibodies that are conjugated to a fluorochrome. Indirect detection, however, uses an unlabeled primary antibody targeting the molecule of interest and a labeled secondary antibody that is directed against the constant region of the primary antibody.</p> <p><strong>Which Detection Method is Better – Direct or Indirect?</strong></p> <p>The optimal detection method may vary from experiment to experiment. The factors and considerations listed in the table below can help determine which method is best suited for your experiment.</p> <figure class="responsive-figure-table"><table class="table table-striped"> <thead> <tr> <th class="bt-blue-table-header"> </th> <th class="bt-blue-table-header"><strong>Direct Detection Method</strong></th> <th class="bt-blue-table-header"><strong>Indirect Detection Method</strong></th> </tr> </thead> <tbody> <tr> <th>Sensitivity</th> <td>The direct method of detection is less sensitive because the amplification of signal afforded by the secondary antibody is lost.</td> <td>The indirect method of detection is highly sensitive as more than one secondary antibody can bind to a single primary antibody molecule.</td> </tr> <tr> <th><strong>Assay Time</strong></th> <td>The direct method protocol is shorter due to the elimination of the secondary antibody incubation steps.</td> <td>The indirect method protocol is longer due to secondary antibody incubation and the resistent wash steps.</td> </tr> <tr> <th><strong>Multiplexing</strong></th> <td>Multiplexing is easier with direct detection because multiple primary antibodies from the same host species can be used together.</td> <td>Indirect method multiplexing is more complicated due to secondary antibody cross-reactivity.</td> </tr> <tr> <th><strong>Background</strong></th> <td>Nonspecific background signal is lower in the direct method as secondary antibody mediated cross-reactivity is eliminated.</td> <td>Nonspecific background staining is generally higher in the indirect method due to secondary antibody cross-reactivity.</td> </tr> <tr> <th><strong>Flexibility</strong></th> <td>The direct method is less flexible since the selection of directly conjugated antibodies can be limited.</td> <td>The indirect method is more flexible because it is possible to use different conjugated secondary antibodies depending on your experiment.</td> </tr> </tbody> </table></figure></div> </div> </div> </div> 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