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<title>Application of anti-glycosaminoglycan antibody and biotinylated hyaluronan binding protein (bHABP) [3] ~ Immunostaining using anti-heparan sulfate antibody - Glycoscience Protocols (GlycoPODv2) - NCBI Bookshelf</title>
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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Glycoscience Protocols (GlycoPODv2) [Internet]" /><meta name="citation_title" content="Application of anti-glycosaminoglycan antibody and biotinylated hyaluronan binding protein (bHABP) [3] ~ Immunostaining using anti-heparan sulfate antibody" /><meta name="citation_publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="citation_date" content="2022/03/30" /><meta name="citation_author" content="Shuhei Yamada" /><meta name="citation_pmid" content="37590702" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK593971/" /><meta name="citation_keywords" content="heparan sulfate" /><meta name="citation_keywords" content="immunostaining" /><meta name="citation_keywords" content="antibody" /><meta name="citation_keywords" content="cellular localization" /><meta name="citation_keywords" content="tissue distribution" /><meta name="citation_keywords" content="proteoglycan" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Application of anti-glycosaminoglycan antibody and biotinylated hyaluronan binding protein (bHABP) [3] ~ Immunostaining using anti-heparan sulfate antibody" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="DC.Contributor" content="Shuhei Yamada" /><meta name="DC.Date" content="2022/03/30" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK593971/" /><meta name="description" content="HS is ubiquitously expressed on the cell surface and in the extracellular matrix of animal tissues. Immunostaining using anti-HS antibodies is an effective way for visualizing the cellular localization and tissue distribution of HS. Although the reactivity of HS to an anti-HS antibody differs depending on the epitope content along the HS chain, here, we show the protocols of immunostaining COS-7 cells and a liver tissue with an anti-HS antibody, F58-10E4, as examples." /><meta name="og:title" content="Application of anti-glycosaminoglycan antibody and biotinylated hyaluronan binding protein (bHABP) [3] ~ Immunostaining using anti-heparan sulfate antibody" /><meta name="og:type" content="book" /><meta name="og:description" content="HS is ubiquitously expressed on the cell surface and in the extracellular matrix of animal tissues. Immunostaining using anti-HS antibodies is an effective way for visualizing the cellular localization and tissue distribution of HS. Although the reactivity of HS to an anti-HS antibody differs depending on the epitope content along the HS chain, here, we show the protocols of immunostaining COS-7 cells and a liver tissue with an anti-HS antibody, F58-10E4, as examples." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK593971/" /><meta name="og:site_name" content="NCBI Bookshelf" /><meta name="og:image" content="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-glycopodv2-lrg.png" /><meta name="twitter:card" content="summary" /><meta name="twitter:site" content="@ncbibooks" /><meta name="bk-non-canon-loc" content="/books/n/glycopodv2/g161-antiglycosamin3/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK593971/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} </style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script>
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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-. </p></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK593971_"><span class="title" itemprop="name">Application of anti-glycosaminoglycan antibody and biotinylated hyaluronan binding protein (bHABP) [3] ~ Immunostaining using anti-heparan sulfate antibody</span></h1><div class="contrib half_rhythm"><span itemprop="author">Shuhei Yamada</span>, Ph.D.<div class="affiliation small">Meijo Univ.<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-ojiem@yiehuhs" class="oemail">pj.ca.u-ojiem@yiehuhs</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">October 4, 2021</span>; Last Revision: <span itemprop="dateModified">March 30, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g161-antiglycosamin3.Introduction"><h2 id="_g161-antiglycosamin3_Introduction_">Introduction</h2><p>The cellular localization and tissue distribution of heparan sulfate (HS) can be investigated by immunostaining using anti-HS antibodies (<a class="bk_pop" href="#g161-antiglycosamin3.REF.1">1</a>–<a class="bk_pop" href="#g161-antiglycosamin3.REF.3">3</a>). However, HS preparations derived from various sources differ in their reactivity to an anti-HS antibody depending on the epitope content along the HS chain. A single anti-HS antibody can react with only particular HS chains, which have a specific epitope structure. To detect all HS-proteoglycans simultaneously, the anti-HS “stub” antibody, F69-3G10, is available after treatment with bacterial heparin/HS lyases. The epitope structure of this antibody is generated only by digestion with the bacterial lyases. Characteristics of major anti-HS antibodies are summarized in the chapter “Application of anti-glycosaminoglycan antibodies and biotinylated hyaluronan binding protein (bHABP) [1] ~ Characteristics and epitopes for anti-heparan sulfate antibodies.”</p></div><div id="g161-antiglycosamin3.Protocol"><h2 id="_g161-antiglycosamin3_Protocol_">Protocol</h2><p>HS is ubiquitously expressed on the cell surface and in the extracellular matrix of animal tissues. Immunostaining using anti-HS antibodies is an effective way for visualizing the cellular localization and tissue distribution of HS. Although the reactivity of HS to an anti-HS antibody differs depending on the epitope content along the HS chain, here, we show the protocols of immunostaining COS-7 cells and a liver tissue with an anti-HS antibody, F58-10E4, as examples.</p><div id="g161-antiglycosamin3.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">0.01 M Phosphate-buffered saline, pH 7.4 (PBS)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Paraformaldehyde (PFA)</p></dd><dt>3.</dt><dd><p class="no_top_margin">Bovine serum albumin (BSA)</p></dd><dt>4.</dt><dd><p class="no_top_margin">Anti-HS antibody, F58-10E4 (Seikagaku Corp., Tokyo, Japan) (<b>Note 1</b>)</p></dd><dt>5.</dt><dd><p class="no_top_margin">Alexa Fluor<sup>®</sup> 488 goat anti-mouse IgM, 2 mg/mL (Thermo Fisher Scientific, Waltham, MA, USA)</p></dd><dt>6.</dt><dd><p class="no_top_margin">Anti-mouse IgM biotin conjugate antibody</p></dd><dt>7.</dt><dd><p class="no_top_margin">4’,6-Diamino-2-phenylindole (DAPI) (Thermo Fisher Scientific)</p></dd><dt>8.</dt><dd><p class="no_top_margin">VECTASHIELS mounting medium (Vector Laboratories Inc., Burlingame, CA)</p></dd><dt>9.</dt><dd><p class="no_top_margin">30% H<sub>2</sub>O<sub>2</sub></p></dd><dt>10.</dt><dd><p class="no_top_margin">Vectastain ABC kit (Vector Laboratories Inc.)</p></dd><dt>11.</dt><dd><p class="no_top_margin">3-3’-Diaminobenzidine (DAB) tetrahydrochloride</p></dd></dl></div><div id="g161-antiglycosamin3.Instrument"><h3>Instrument</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Fluorescence microscope, BZ-9000 (KEYENCE Corporation, Osaka, Japan)</p></dd></dl></div><div id="g161-antiglycosamin3.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Staining of cells</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Seed cells on a cover glass in a 6-cm dish.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Transfer the cover glass to a 6-well plate and wash it with PBS three times.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Add 1 mL of 4% PFA/PBS to the well for fixing and incubate at room temperature for 30 min.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Wash the cover glass with PBS for 1 s once and then for 5 min three times. (<b>Note 2</b>)</p></dd><dt>e.</dt><dd><p class="no_top_margin">Add 200 μL of 1% BSA/PBS to the cover glass for blocking and incubate at room temperature for 30 min.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Wash the cover glass with PBS twice and then with 1% BSA/PBS once.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Transfer the cover glass to a humid box.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Add 200 μL of the anti-HS antibody F58-10E4 (diluted 1:200 in 1% BSA/PBS) and incubate at 4°C overnight. (<b>Note 3</b>)</p></dd><dt>i.</dt><dd><p class="no_top_margin">Transfer the cover glass to a 6-well plate, and wash it with PBS for 5 min three times.</p></dd><dt>j.</dt><dd><p class="no_top_margin">Wash the cover glass with 1% BSA/PBS twice.</p></dd><dt>k.</dt><dd><p class="no_top_margin">Transfer the cover glass to a humid box.</p></dd><dt>l.</dt><dd><p class="no_top_margin">Add 200 μL of the secondary antibody, Alexa Fluor® 488 goat anti-mouse IgM (diluted 1: 200 in 1% BSA/PBS), and incubate for 1 h at room temperature with light shielding (<b>Note 3</b>).</p></dd><dt>m.</dt><dd><p class="no_top_margin">Transfer the cover glass to a 6-well plate and wash it with PBS for 5 min three times.</p></dd><dt>n.</dt><dd><p class="no_top_margin">Add 1 mL of 5 ng/mL DAPI and incubate for 5 min at room temperature with light shielding.</p></dd><dt>o.</dt><dd><p class="no_top_margin">Wash the cover glass with PBS for 1 s once and then for 5 min three times.</p></dd><dt>p.</dt><dd><p class="no_top_margin">Mount the cover glass using the mounting medium.</p></dd><dt>q.</dt><dd><p class="no_top_margin">Capture images using a fluorescent microscope (<a class="figpopup" href="/books/NBK593971/figure/g161-antiglycosamin3.F1/?report=objectonly" target="object" rid-figpopup="figg161antiglycosamin3F1" rid-ob="figobg161antiglycosamin3F1">Figure 1</a>).</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Staining of tissues</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Soak a paraffin-embedded section on a glass slide in xylene (three times), ethanol (twice), 90%, 70%, and 50% ethanol sequentially for 5 min each, and then in 2 changes of H<sub>2</sub>O for 5 min each in a Coplin jar at room temperature for deparaffinization and hydration (<b>Note 4</b>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Soak the section in two changes in PBS for 5 min and then 0.1% Triton X-100/PBS for 10 min for the retrieval of the antigen.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Soak the slide in two changes in PBS and then in H<sub>2</sub>O for 5 min.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Soak the slide in 3% H<sub>2</sub>O<sub>2</sub>/methanol at room temperature for 30 min for the inactivation of endogenous peroxidases.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Soak the slide in two changes in H<sub>2</sub>O and then two changes in PBS for 5 min each.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Add 200 μL of 3% BSA/PBS to the section for blocking in a humid box and incubate for 1 h at room temperature.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Tap off the excess BSA/PBS solution.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Add 200 μL of the anti-HS antibody, F58-10E4 (diluted 1:100 in 1% BSA/PBS), to the section in a humid box and incubate at 4°C overnight.</p></dd><dt>i.</dt><dd><p class="no_top_margin">Tap off the excess antibody solution.</p></dd><dt>j.</dt><dd><p class="no_top_margin">Soak the slide in PBS for 1 s once and then in three changes in PBS for 5 min each (<b>Note 5</b>).</p></dd><dt>k.</dt><dd><p class="no_top_margin">Add 200 μL of the secondary antibody, anti-mouse IgM biotin conjugate antibody (diluted 1:100 in 1% BSA/PBS), to the section in a humid box and incubate at room temperature for 1 h.</p></dd><dt>l.</dt><dd><p class="no_top_margin">Tap off the excess secondary antibody solution.</p></dd><dt>m.</dt><dd><p class="no_top_margin">Soak the slide in PBS for 1 s once and then in three changes in PBS for 5 min each.</p></dd><dt>n.</dt><dd><p class="no_top_margin">Add 200 μL of the ABC solution of the Vectastain ABC kit (diluted 1:200 in PBS) to the section in a humid box and incubate at room temperature for 1 h.</p></dd><dt>o.</dt><dd><p class="no_top_margin">Tap off the excess ABC solution.</p></dd><dt>p.</dt><dd><p class="no_top_margin">Soak the slide in PBS for 1 s once and then in three changes in PBS for 5 min each (<b>Note 5</b>)<b>.</b></p></dd><dt>q.</dt><dd><p class="no_top_margin">Soak the slide in 0.06% DAB/PBS for 10 min and then in 0.01% H<sub>2</sub>O<sub>2</sub>/0.06% DAB/PBS within 10 min until the color is developed.</p></dd><dt>r.</dt><dd><p class="no_top_margin">Soak the slide in PBS for 1 s to terminate the reaction and then in three changes in PBS for 5 min each.</p></dd><dt>s.</dt><dd><p class="no_top_margin">Soak the slide in H<sub>2</sub>O for 5 min and then in 20%, 40%, 80%, and 100% ethanol sequentially for 5 min each.</p></dd><dt>t.</dt><dd><p class="no_top_margin">Soak the slide in three changes in xylene for 10 min each.</p></dd><dt>u.</dt><dd><p class="no_top_margin">Dry the section in air.</p></dd><dt>v.</dt><dd><p class="no_top_margin">Mount the sections under a cover glass using the mounting medium.</p></dd><dt>w.</dt><dd><p class="no_top_margin">Capture images using a microscope (<a class="figpopup" href="/books/NBK593971/figure/g161-antiglycosamin3.F2/?report=objectonly" target="object" rid-figpopup="figg161antiglycosamin3F2" rid-ob="figobg161antiglycosamin3F2">Figure 2</a>).</p></dd></dl></dd></dl></div><div id="g161-antiglycosamin3.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Seikagaku Corp. discontinued its research reagent business on Sept. 30, 2011. Anti-HS antibodies are available from several companies, including Cosmo Bio Co., Ltd., Tokyo, Japan. Authentic HS preparations can be purchased from Sigma-Aldrich.</p></dd><dt>2.</dt><dd><p class="no_top_margin">To permeabilize the cells, they are treated with the lysis buffer (PBS containing 0.1% Triton X-100) for 5 min at room temperature after step 1d of Methods and then washed with PBS twice.</p></dd><dt>3.</dt><dd><p class="no_top_margin">The antibody solution should be diluted just before use.</p></dd><dt>4.</dt><dd><p class="no_top_margin">In the case of a frozen section, the section is soaked in acetone/methanol (1:1) for fixing and incubated at room temperature for 3 min. After the section has been dried in air, experiments can be performed from step 2d of Methods.</p></dd><dt>5.</dt><dd><p class="no_top_margin">Be careful not to overdevelop the preparations. The incubation period can be changed.</p></dd></dl></div></div><div id="g161-antiglycosamin3.References"><h2 id="_g161-antiglycosamin3_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g161-antiglycosamin3.REF.1">Li J, Kleeff J, Abiatari I, Kayed H, Giese NA, Felix K, Giese T, Büchler MW, Friess H. Enhanced levels of Hsulf-1 interfere with heparin-binding growth factor signaling in pancreatic cancer. <span><span class="ref-journal">Mol Cancer. </span>2005 Apr 7;<span class="ref-vol">4</span>(1):14.</span> [<a href="/pmc/articles/PMC1087876/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC1087876</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/15817123" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 15817123</span></a>] [<a href="http://dx.crossref.org/10.1186/1476-4598-4-14" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g161-antiglycosamin3.REF.2">Stickens D, Zak BM, Rougier N, Esko JD, Werb Z. Mice deficient in Ext2 lack heparan sulfate and develop exostoses. <span><span class="ref-journal">Development. </span>2005 Nov;<span class="ref-vol">132</span>(22):5055–68.</span> [<a href="/pmc/articles/PMC2767329/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC2767329</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/16236767" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16236767</span></a>] [<a href="http://dx.crossref.org/10.1242/dev.02088" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g161-antiglycosamin3.REF.3">Pan Y, Woodbury A, Esko JD, Grobe K, Zhang X. Heparan sulfate biosynthetic gene Ndst1 is required for FGF signaling in early lens development. <span><span class="ref-journal">Development. </span>2006 Dec;<span class="ref-vol">133</span>(24):4933–44.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17107998" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17107998</span></a>] [<a href="http://dx.crossref.org/10.1242/dev.02679" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593971_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g161-antiglycosamin3.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div class="bk_prnt_sctn"><h2>Figures</h2><div class="whole_rhythm bk_prnt_obj bk_first_prnt_obj"><div id="g161-antiglycosamin3.F1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201%3A%20.%20The%20immunostaining%20of%20COS-7%20cells%20for%20heparan%20sulfate%20using%20F58-10E4.&p=BOOKS&id=593971_g161-antiglycosamin3-Image001.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK593971/bin/g161-antiglycosamin3-Image001.jpg" alt="Figure 1: . The immunostaining of COS-7 cells for heparan sulfate using F58-10E4." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>The immunostaining of COS-7 cells for heparan sulfate using F58-10E4.</p><p>Strong staining (green) was observed at the periphery of the cells. 4’,6-Diamino-2-phenylindole stains the nucleus (blue). Scale bar, 50 μm.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g161-antiglycosamin3.F2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593971/bin/g161-antiglycosamin3-Image002.jpg" alt="Figure 2: . Sections of liver stained with the anti–heparan sulfate antibody F58-10E4." /></div><h3><span class="label">Figure 2: </span></h3><div class="caption"><p>Sections of liver stained with the anti–heparan sulfate antibody F58-10E4.</p><p>A significant staining with F58-10E4 (A) was observed between hepatic cells (indicated by arrows) but not with normal mouse IgM (negative control) (B). Scale bar, 50 μm.</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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