{"id":9154,"date":"2022-06-17T09:35:42","date_gmt":"2022-06-17T13:35:42","guid":{"rendered":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/?p=9154"},"modified":"2025-03-18T11:08:04","modified_gmt":"2025-03-18T15:08:04","slug":"focus-imaging-with-affinipure-vhh-secondary-antibodies","status":"publish","type":"post","link":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/trending-topics\/focus-imaging-with-affinipure-vhh-secondary-antibodies\/","title":{"rendered":"Focus: Imaging with AffiniPure-VHH\u00ae Secondary Antibodies"},"content":{"rendered":"\n\n\n\t<div class=\"dkpdf-button-container\" style=\" text-align:right \">\n\n\t\t<a class=\"dkpdf-button\" href=\"\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/9154?pdf=9154\" target=\"_blank\"><span class=\"dkpdf-button-icon\"><i class=\"fa fa-file-pdf-o\"><\/i><\/span> Download PDF<\/a>\n\n\t<\/div>\n\n\n\n\n\n<style>.entry p, .entry ol{font-size: 1rem;}.entry h3{color:#009fe3;margin-top:0}.entry h4{color:#009453;margin-top:1.5rem}.entry h5{color: #003a5c; font-size: 1.05rem;margin-top:1.5rem;}.entry figure{margin:32px auto;border:1px solid #ccc}.entry figure img{width:100%;display:block;margin:0 auto}.entry figcaption{font-size:.875rem;line-height:1.35rem;padding:10px;color:#222}.entry .blog-tbl{margin:1rem auto;caption-side:bottom}.entry .blog-tbl td,.entry .blog-tbl th{padding:10px 9px;border:1px solid #00172b;text-align:left}.entry .blog-tbl td{border-color:#003a5c}.entry .blog-tbl th{background-color:#00172b;color:#fff;border-left-color:#fff;border-right-color:#fff}.entry .blog-tbl th p{color:#fff}.entry .blog-tbl th:first-of-type{border-left-color:#00172b}.entry .blog-tbl th:last-of-type{border-right-color:#00172b}.entry .blog-tbl p{text-align:left;margin:0}.entry .box-note{border:2px solid #009453;padding:12px;margin:1rem 0}.entry .box-note p{margin:0;padding:0}.entry .styled-list{list-style-type:none}.entry .styled-list li{margin-top:1rem;line-height:1rem;font-size:1rem;}.entry .styled-list li::before{font-family:\"Font Awesome 5 Pro\";display:inline-block;content:\"\\f3c5\";-webkit-transform:rotate(-90deg);transform:rotate(-90deg);margin-left:-20px;margin-right:11px;font-size:.75rem;color:#ed7004;font-weight:600}.entry .styled-list ol li::before{display:none}.entry .styled-list li>ul li::before{font-weight:200}.entry .overview{width:-webkit-fit-content;width:-moz-fit-content;width:fit-content;padding:16px;margin:1rem auto;border:1px solid #eee}.entry .overview hr{margin-top:14px}.entry .overview-text{text-align:center;font-size:1rem;margin:0}.entry .btn-sq{color:#fff;font-size:.9rem;font-weight:600;border:2px solid rgb(237, 112, 4);padding:7px 13px;background:rgb(237, 112, 4);cursor:pointer;text-align:center;line-height:1.5rem;margin:0 auto;}.entry .btn-sq:hover{text-decoration:none;color:rgb(237, 112, 4);background:#fff;}.entry .btn-container{display:flex;width:100%;margin:1.3rem 0;}.entry .btn-container .fa-solid, .entry .btn-container .fa-duotone{margin-right:10px;}@media(max-width: 768px){.entry .btn-sq{font-size:1.05rem;}}<\/style>\n<style>.entry figure {max-width: 550px}.vertical-btns{flex-direction:column;}.vertical-btns .btn-sq{max-width:88%;width:490px}.vertical-btns .btn-sq:last-of-type{margin-top:1rem;}<\/style>\n<p><script src=\"https:\/\/kit.fontawesome.com\/904923013f.js\" crossorigin=\"anonymous\"><\/script><\/p>\n<h2><span style=\"font-weight: 400;\">Combining the exquisite specificity our minimally cross-reactive antibodies have always offered with the unique penetrative capabilities of the small VHH fragment format, JIR AffiniPure-VHH\u2122 Secondary Antibodies enable high precision staining. Read our article to explore how they behave in mouse brain tissue.&nbsp;<\/span><\/h2>\n<h4>Staining brain tissue<\/h4>\n<p><span style=\"font-weight: 400;\">Staining any tissue requires experimental considerations; sometimes, it\u2019s about the potential for cross-reactivity, sometimes it\u2019s the permeability of the reagents, and sometimes it\u2019s about finding the right reporter molecule. When staining multiple targets, all three considerations are critical to generating results that illustrate clear, impactful, and visually appealing findings. Whether you want to determine protein expression, where it\u2019s localized, or if it colocalizes with another molecule, JIR AffiniPure-VHH\u2122 Secondary Antibodies can help you amplify the detail.&nbsp;<\/span><\/p>\n<figure><img decoding=\"async\" loading=\"lazy\" width=\"1063\" height=\"881\" class=\"aligncenter wp-image-9219 size-full\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Brain-IF-multilabel-single-dilution-edit.jpg\" alt=\"\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Brain-IF-multilabel-single-dilution-edit.jpg 1063w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Brain-IF-multilabel-single-dilution-edit-300x249.jpg 300w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/Brain-IF-multilabel-single-dilution-edit-1024x849.jpg 1024w\" sizes=\"(max-width: 1063px) 100vw, 1063px\" \/><figcaption><strong>Figure 1: Immunohistochemistry for Tyrosine Hydroxylase (green) and NeuN (red) in formalin-fixed paraffin-embedded (FFPE) mouse brain.<\/strong><br \/>\nSagittal sections of mouse brain were blocked with 5% normal alpaca serum in Tris buffer. Primary antibodies were applied at 4C overnight at the following dilutions: rabbit anti-Tyrosine Hydroxylase (Abcam #ab112) at a 1:250 dilution; mouse anti-NeuN (Millipore #MAB377) at a 1:100 dilution. The following secondary antibodies were applied at room temperature for one hour at 1:220 dilutions: Alexa Fluor\u00ae 488 VHH Fragment Alpaca Anti-Rabbit IgG (H+L) (<a href=\"\/catalog\/products\/611-544-215\">611-544-215<\/a>); Cy3 VHH Fragment Alpaca Anti-Mouse IgG (H+L) (<a href=\"\/catalog\/products\/615-164-214\">615-164-214<\/a>). Nuclei of cells were visualized using DAPI (blue). Images were obtained from slides created and provided by UNC\u2019s Histology Research Core Facility.<\/figcaption><\/figure>\n<hr>\n<h4>Polyclonal antibodies let you amplify the detail<\/h4>\n<p><span style=\"font-weight: 400;\">Polyclonal AffiniPure-VHH\u2122 Secondary Antibodies offer excellent sensitivity through signal amplification. As seen in Figure 2, polyclonal antibodies achieve higher labeling efficiency than monoclonal antibodies by binding to multiple locations across the primary antibody, generating a brighter signal.<\/span><\/p>\n<figure><img decoding=\"async\" loading=\"lazy\" class=\"aligncenter wp-image-10822\" src=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/JIR-AffiniPure-VHH\u2122-poly-mono-Illustration-1.jpg\" alt=\"\" width=\"600\" height=\"318\" srcset=\"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/JIR-AffiniPure-VHH\u2122-poly-mono-Illustration-1.jpg 936w, https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-content\/uploads\/JIR-AffiniPure-VHH\u2122-poly-mono-Illustration-1-300x159.jpg 300w\" sizes=\"(max-width: 600px) 100vw, 600px\" \/><figcaption><strong>Figure 2.<\/strong> Schematic showing the increased number of conjugated polyclonal AffiniPure-VHH\u2122 Secondary Antibodies able to bind a primary antibody as compared to monoclonal nanobodies. Also, the diameter of the complex remains comparable, with the distance between the probe and target protein being similar.<\/figcaption><\/figure>\n<hr>\n<h4>Min-X for exquisite differentiation<\/h4>\n<p><span style=\"font-weight: 400;\">When visualizing multiple targets, cross-adsorbed antibodies allow you to build up a multicolored image without worrying about the off-target signal from cross-reacting antibodies.&nbsp; AffiniPure-VHH\u2122 Secondary Antibodies are available cross-adsorbed (min X) to commonly used species which can reduce background and enhance specificity, enabling you to experience exquisite differentiation of target proteins.<\/span><\/p>\n<div class=\"btn-container vertical-btns\"><a class=\"btn-sq\" href=\"\/technical\/products\/cross-adsorbed-secondary-antibodies\"><i class=\"fa-duotone fa-book-open-reader\"><\/i>Read more about cross-adsorbed antibodies<\/a><br \/>\n<a class=\"btn-sq\" href=\"\/technical\/products\/protocols\/multiple-labeling\"><i class=\"fa-duotone fa-book-open-reader\"><\/i>Read more about designing multiple labeling experiments<\/a><\/div>\n<hr>\n<h4>Small means better penetration<\/h4>\n<p>One-tenth the size of conventional antibodies, these tiny antibody fragments offer excellent tissue penetration allowing you to reduce incubations or access targets that require specialized permeabilization treatment. Nanobodies lack an Fc effector domain which allows enhanced clearance and aids live-cell imaging techniques such as Immuno-PET.<\/p>\n<div class=\"btn-container\"><a class=\"btn-sq\" href=\"\/technical\/products\/AffiniPure-VHH-secondary-antibodies\"><i class=\"fa-duotone fa-book-open-reader\"><\/i>Read more about the advantages of AffiniPure-VHH\u2122 Secondary Antibodies<\/a><\/div>\n<hr>\n<h4>Better choice &#8211; choose from the whole spectrum<\/h4>\n<p><span style=\"font-weight: 400;\">Don&#8217;t be limited by conjugate availability; when a directly conjugated antibody isn\u2019t available, JIR AffiniPure-VHH\u2122 Secondary Antibodies give you access to a range of dyes without compromising resolution. We offer DyLight, Alexa Fluor\u00ae, and Cyanine\u2122 dyes covering the spectrum, so you can choose suitable conjugates for your filter set.&nbsp;<\/span><\/p>\n<div class=\"btn-container\"><a class=\"btn-sq\" href=\"\/technical\/products\/AffiniPure-VHH-secondary-antibodies\"><i class=\"fa-solid fa-graduation-cap\"><\/i>Learn more about JIR AffiniPure-VHH\u2122 Secondary Antibodies<\/a><\/div>\n<hr>\n<h4>References:<\/h4>\n<ul class=\"styled-list\">\n<li aria-level=\"1\">Carrington, G., Tomlinson, D. and Peckham, M., 2019. Exploiting nanobodies and Affimers for superresolution imaging in light microscopy. Molecular Biology of the Cell, 30(22), pp.2737-2740.<\/li>\n<li aria-level=\"1\">Chakravarty, R., Goel, S., and Cai, W., 2014. Nanobody: The \u201cMagic Bullet\u201d for Molecular Imaging?. Theranostics, 4(4), pp.386-398.<\/li>\n<li aria-level=\"1\">De Groeve, K., Deschacht, N., De Koninck, C., Caveliers, V., Lahoutte, T., Devoogdt, N., Muyldermans, S., De Baetselier, P. and Raes, G., 2010. Nanobodies as Tools for In Vivo Imaging of Specific Immune Cell Types. Journal of Nuclear Medicine, 51(5), pp.782-789.<\/li>\n<li aria-level=\"1\">Debie, P., Lafont, C., Defrise, M., Hansen, I., van Willigen, D., van Leeuwen, F., Gijsbers, R., D&#8217;Huyvetter, M., Devoogdt, N., Lahoutte, T., Mollard, P. and Hernot, S., 2020. Size and affinity kinetics of nanobodies influence targeting and penetration of solid tumours. Journal of Controlled Release, 317, pp.34-42.<\/li>\n<li aria-level=\"1\">Erreni, M., Schorn, T., D\u2019Autilia, F. and Doni, A., 2020. Nanobodies as Versatile Tool for Multiscale Imaging Modalities. Biomolecules, 10(12), p.1695.<\/li>\n<li aria-level=\"1\">Hassanzadeh-Ghassabeh, G., Devoogdt, N., De Pauw, P., Vincke, C. and Muyldermans, S., 2013. Nanobodies and their potential applications. Nanomedicine, 8(6), pp.1013-1026.<\/li>\n<li aria-level=\"1\">Jov\u010devska, I. and Muyldermans, S., 2019. The Therapeutic Potential of Nanobodies. BioDrugs, 34(1), pp.11-26.<\/li>\n<li aria-level=\"1\">Krasniqi, A., D\u2019Huyvetter, M., Devoogdt, N., Frejd, F., S\u00f6rensen, J., Orlova, A., Keyaerts, M. and Tolmachev, V., 2018. Same-Day Imaging Using Small Proteins: Clinical Experience and Translational Prospects in Oncology. Journal of Nuclear Medicine, 59(6), pp.885-891.<\/li>\n<li aria-level=\"1\">Stark, H. Urlaub, and D. G\u00f6rlich. 2015. Nanobodies: Site-specific labeling for super-resolution imaging, rapid epitope-mapping, and native protein complex isolation. eLife. 4:e11349. https:\/\/doi.org\/10.7554\/eLife.11349<\/li>\n<\/ul>\n<p><a href=\"https:\/\/www.jacksonimmuno.com\/technical\/products\/nano-secondary-antibodies?utm_campaign=Q2%20promo%20Nano%20Secondaries&amp;utm_source=Blogbanner&amp;utm_medium=Blogbanner\"><img decoding=\"async\" loading=\"lazy\" class=\"aligncenter wp-image-9179 size-full\" src=\"https:\/\/www.jacksonimmuno.com\/img\/static-pages\/AffiniPure-VHH_banner.jpg\" alt=\"\" width=\"728\" height=\"90\"><\/a><\/p>\n<table class=\"table blogLinks aligncenter\">\n<thead>\n<tr>\n<th class=\"span6\">Learn more:<\/th>\n<th class=\"span6\">Do more:<\/th>\n<\/tr>\n<\/thead>\n<tbody>\n<tr>\n<td class=\"span6\"><a href=\"\/secondary-antibody-resource\/immuno-techniques\/colorimetric-western-blotting\/\">Colorimetric western blotting<\/a><\/td>\n<td class=\"span6\"><strong><a href=\"https:\/\/www.jacksonimmuno.com\/technical\/products\/conjugate-selection\/fluorophore-selection#spectra\">Spectra Viewer<\/a><\/strong><\/td>\n<\/tr>\n<tr>\n<td class=\"span6\"><a href=\"\/secondary-antibody-resource\/immuno-techniques\/fluorescent-western-blotting\/\">Chemiluminescence western blotting<\/a><\/td>\n<td class=\"span6\"><a href=\"\/technical\/products\/groups\/signal-enhancement\">Antibodies for signal enhancement<\/a><\/td>\n<\/tr>\n<tr>\n<td class=\"span6\" colspan=\"2\"><a href=\"\/secondary-antibody-resource\/immuno-techniques\/fluorescent-western-blotting\/\">Fluorescent western blotting<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<!-- AddThis Advanced Settings generic via filter on the_content --><!-- AddThis Share Buttons generic via filter on the_content --><!-- AddThis Related Posts generic via filter on the_content -->","protected":false},"excerpt":{"rendered":"<p>Download PDF Combining the exquisite specificity our minimally cross-reactive antibodies have always offered with the unique penetrative capabilities of the small VHH fragment format, JIR AffiniPure-VHH\u2122 Secondary Antibodies enable high precision staining. Read our article to explore how they behave in mouse brain tissue.&nbsp; Staining brain tissue Staining any tissue requires experimental considerations; sometimes, it\u2019s [&hellip;]<!-- AddThis Advanced Settings generic via filter on get_the_excerpt --><!-- AddThis Share Buttons generic via filter on get_the_excerpt --><!-- AddThis Related Posts generic via filter on get_the_excerpt --><\/p>\n","protected":false},"author":3,"featured_media":9219,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"content-type":""},"categories":[20],"tags":[],"acf":[],"_links":{"self":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/9154"}],"collection":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/comments?post=9154"}],"version-history":[{"count":39,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/9154\/revisions"}],"predecessor-version":[{"id":11246,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/posts\/9154\/revisions\/11246"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/media\/9219"}],"wp:attachment":[{"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/media?parent=9154"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/categories?post=9154"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.jacksonimmuno.com\/secondary-antibody-resource\/wp-json\/wp\/v2\/tags?post=9154"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}