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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="_NBK593826_"><span class="title" itemprop="name">Anti-chondroitin sulfate antibodies and their utility for immunostaining analyses</span></h1><div class="contrib half_rhythm"><span itemprop="author">Tadahisa Mikami</span>, Ph.D.<div class="affiliation small">Kobe Pharmaceutical Univ.<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-amrahpebok@imakimt" class="oemail">pj.ca.u-amrahpebok@imakimt</a></div></div></div><div class="contrib half_rhythm"><span itemprop="author">Hiroshi Kitagawa</span>, Ph.D.<div class="affiliation small">Kobe Pharmaceutical Univ.<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-amrahpebok@awagatik" class="oemail">pj.ca.u-amrahpebok@awagatik</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">September 22, 2021</span>; Last Revision: <span itemprop="dateModified">January 12, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g165-antichondrsulf.Introduction"><h2 id="_g165-antichondrsulf_Introduction_">Introduction</h2><p>Chondroitin sulfate proteoglycans (CSPGs) are a major class of extra/pericellular matrix macromolecules that not only serve as structural elements to form specialized cellular microenvironments but also guide myriad cellular processes, such as migration, proliferation, differentiation, survival, homeostasis, and regeneration (<a class="bk_pop" href="#g165-antichondrsulf.REF.1">1</a>–<a class="bk_pop" href="#g165-antichondrsulf.REF.3">3</a>). Usually, such multiple regulatory functions of CSPGs are exerted through the chondroitin sulfate (CS) moieties, whereas their protein cores may primarily play a scaffolding role to assist molecular interplay between CS chains and potential interactors, including cytokines, morphogens, and cell surface receptors (<a class="bk_pop" href="#g165-antichondrsulf.REF.1">1</a>–<a class="bk_pop" href="#g165-antichondrsulf.REF.3">3</a>). Since expression status of the glycosaminoglycan (GAG) chains of CSPGs is spatiotemporally regulated by biosynthetic machineries, it is essential to detect the functional expression of CSPGs in certain cell culture system and tissue sections to understand the biological roles of CSPGs.</p></div><div id="g165-antichondrsulf.Protocol"><h2 id="_g165-antichondrsulf_Protocol_">Protocol</h2><p>This section describes the basic protocols for immunocytochemical and immunohistochemical detection of CS chains of CSPGs. For a summary of commercially available anti-CS antibodies, please refer the chapter “Anti-chondroitin sulfate antibodies and their utility for enzyme-linked immunosorbent assay and western blotting”.</p><div id="g165-antichondrsulf.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Chondroitinase ABC (<i>Proteus vulgaris</i>, Seikagaku, Tokyo, Japan) (<b>Notes 1</b> and <b>2</b>)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Anti-CS monoclonal antibody (2B6, mouse IgG<sub>1</sub>, Cosmo Bio, Tokyo, Japan)</p></dd><dt>3.</dt><dd><p class="no_top_margin">Anti-CS monoclonal antibody (CS-56, mouse IgM, Sigma-Aldrich, St. Louis, MO)</p></dd><dt>4.</dt><dd><p class="no_top_margin">Fluorophore-conjugated antimouse IgG (Thermo Fisher Scientific, Waltham, CA)</p></dd><dt>5.</dt><dd><p class="no_top_margin">Fluorophore-conjugated antimouse IgM (Thermo Fisher Scientific, Waltham, CA)</p></dd><dt>6.</dt><dd><p class="no_top_margin">Nuclear staining reagent [DAPI (4’,6-diamidino-2-phenylindole), or NucBlue™ Fixed Cell Stain ReadyProbes™ reagent, Thermo Fisher Scientific, Waltham, CA]</p></dd><dt>7.</dt><dd><p class="no_top_margin">Mounting medium (Fluoroshield, ImmunoBioScience, Mukilteo, WA)</p></dd><dt>8.</dt><dd><p class="no_top_margin">Phosphate-buffered saline (PBS, Dulbecco’s PBS, Nacalai Tesque, Kyoto, Japan)</p></dd><dt>9.</dt><dd><p class="no_top_margin">4% (w/v) paraformaldehyde (PFA)/PBS, pH 7.4</p></dd><dt>10.</dt><dd><p class="no_top_margin">Methanol</p></dd><dt>11.</dt><dd><p class="no_top_margin">5× chondroitinase reaction buffer (250 mM Tris, 300 mM sodium acetate, pH 8.0)</p></dd><dt>12.</dt><dd><p class="no_top_margin">Bovine serum albumin (BSA, Fujifilm, Osaka, Japan)</p></dd><dt>13.</dt><dd><p class="no_top_margin">3% (w/v) BSA/PBS</p></dd></dl></div><div id="g165-antichondrsulf.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Fluorescence microscope (BZ-X series, Keyence, Osaka, Japan)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Microtome or cryostat</p></dd><dt>3.</dt><dd><p class="no_top_margin">Humidity chamber</p></dd></dl></div><div id="g165-antichondrsulf.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Immunocytochemistry</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Seed and culture cells on a glass chamber slide or a cover glass.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Rinse briefly with PBS.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Fix the cells with 4% PFA/PBS for 10 min at room temperature.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Wash the cells with PBS three times (<b>Note 3</b>).</p></dd><dt>e.</dt><dd><p class="no_top_margin">Incubate a part of the fixed cells at 37˚C for 2 h with 20 mIU of chondroitinase ABC (<b>Notes 4</b> and <b>5</b>).</p></dd><dt>f.</dt><dd><p class="no_top_margin">Wash the cells with PBS three times.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Incubate the cells with 3% (w/v) BSA/PBS at room temperature for 1 h.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Incubate the cells with an anti-CS antibody diluted with 3% (w/v) BSA/PBS, and incubate at 4˚C overnight (<b>Note 6</b>).</p></dd><dt>i.</dt><dd><p class="no_top_margin">Wash the cells with PBS three times.</p></dd><dt>j.</dt><dd><p class="no_top_margin">Incubate the cells with a fluorophore-conjugated secondary antibody diluted 1,000-fold with 3% (w/v) BSA/PBS at room temperature for 1 h.</p></dd><dt>k.</dt><dd><p class="no_top_margin">Wash the cells with PBS three times.</p></dd><dt>l.</dt><dd><p class="no_top_margin">Counterstain the cells with a nuclear staining reagent (DAPI, 0.1–10 µg/ml in PBS) at room temperature for 5–30 min.</p></dd><dt>m.</dt><dd><p class="no_top_margin">Mount the resultant specimens with a mounting medium.</p></dd><dt>n.</dt><dd><p class="no_top_margin">Analyze the specimens under a fluorescence microscope (<a class="figpopup" href="/books/NBK593826/figure/g165-antichondrsulf.F1/?report=objectonly" target="object" rid-figpopup="figg165antichondrsulfF1" rid-ob="figobg165antichondrsulfF1">Figure 1</a>).</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Immunohistochemistry for skeletal muscle sections</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Prepare freshly frozen tissue sections (5–10 µm thickness) using a cryostat (<b>Note 7</b>).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Postfix the sections with 4% PFA/PBS for 10 min at room temperature.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Wash the cells with PBS three times (<b>Note 3</b>).</p></dd><dt>d.</dt><dd><p class="no_top_margin">Incubate a part of the fixed cells at 37˚C for 2 h with 20 mIU of chondroitinase ABC (<b>Notes 4, 5</b> and <b>8</b>).</p></dd><dt>e.</dt><dd><p class="no_top_margin">Incubate the sections with 3% (w/v) BSA/PBS at room temperature for 1 h.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Incubate the sections with an anti-CS antibody diluted with 3% (w/v) BSA/PBS, and incubate at 4˚C overnight (<b>Notes 6</b> and <b>9</b>).</p></dd><dt>g.</dt><dd><p class="no_top_margin">Wash the sections with PBS three times.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Incubate the sections with a fluorophore-conjugated secondary antibody diluted 1,000-fold with 3% (w/v) BSA/PBS at room temperature for 1 h.</p></dd><dt>i.</dt><dd><p class="no_top_margin">Wash the sections with PBS three times.</p></dd><dt>j.</dt><dd><p class="no_top_margin">Counterstain the sections with a nuclear staining reagent (DAPI, 0.1–10 µg/ml in PBS) at room temperature for 5–30 min.</p></dd><dt>k.</dt><dd><p class="no_top_margin">Mount the resultant sections with a mounting medium.</p></dd><dt>l.</dt><dd><p class="no_top_margin">Analyze the sections under a fluorescence microscope (<a class="figpopup" href="/books/NBK593826/figure/g165-antichondrsulf.F2/?report=objectonly" target="object" rid-figpopup="figg165antichondrsulfF2" rid-ob="figobg165antichondrsulfF2">Figure 2</a>).</p></dd></dl></dd></dl></div><div id="g165-antichondrsulf.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Chondroitinase ABC is currently available from Cosmo Bio or Sigma-Aldrich.</p></dd><dt>2.</dt><dd><p class="no_top_margin">Chondroitinase ABC cleaves CS chains by eliminating the GalNAc–GlcA linkages, thereby generating unsaturated disaccharide neoepitopes at nonreducing terminal “CS stub” region of CSPG core protein.</p></dd><dt>3.</dt><dd><p class="no_top_margin">Optional: In some cases, the final immunoreactivity toward CS chains on the specimens may be enhanced by doing additional procedures as follows: [1] After Step d, treat the cells (or sections) with ice-cold methanol at ∆20˚C for 10 min. [2] Remove methanol and air-dry. [3] Incubate the cells (or sections) with PBS for 5 min, and then proceed to Step e. Methanol fixation may be effective at preserving extra/pericellular matrix structures on the specimens but also enhances cell permeability. Thus, be aware of emergence of an undesired intracellular CS immunoreactivity</p></dd><dt>4.</dt><dd><p class="no_top_margin">For immunostaining using anti-CS antibodies (CS-56, etc.) that recognize native CS chains, the chondroitinase-treated preparations serve as a negative control. In contrast, for immunodetection using specific antibodies (1B5, 2B6, and 3B3) that recognize unsaturated CS disaccharide neoepitopes corresponding to the nonreducing terminal “CS stub” region of CSPG core protein, the pretreatment with chondroitinase is essential step.</p></dd><dt>5.</dt><dd><p class="no_top_margin">The reaction mixture should be adjusted to contain one-fifth volume of 5× chondroitinase reaction buffer.</p></dd><dt>6.</dt><dd><p class="no_top_margin">Each researcher must determine the optimum working dilution for each experimental condition.</p></dd><dt>7.</dt><dd><p class="no_top_margin">Perfusion fixation is not suitable for preparation of skeletal muscle tissue sections, due to the emergence of undesired artifacts in the processed sections. In contrast, for many other tissues, sections can be prepared using formalin-fixed, paraffin-embedded samples. If you use paraffin sections, their deparaffinization and rehydration processes are required to proceed to Step b.</p></dd><dt>8.</dt><dd><p class="no_top_margin">In the following steps, use of a humidity chamber is recommended to prevent reaction solution from drying out during immunostaining.</p></dd><dt>9.</dt><dd><p class="no_top_margin">In case of the sections where CS expression level is relatively low, immunostaining aiming at the CS disaccharide neoepitopes may provide a clearer immunoreactivity than that using the anti-CS antibodies recognizing native CS chains.</p></dd></dl></div></div><div id="g165-antichondrsulf.References"><h2 id="_g165-antichondrsulf_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g165-antichondrsulf.REF.1">Mikami T, Kitagawa H. Biosynthesis and function of chondroitin sulfate. <span><span class="ref-journal">Biochim Biophys Acta. </span>2013 Oct;<span class="ref-vol">1830</span>(10):4719–33.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/23774590" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23774590</span></a>] [<a href="http://dx.crossref.org/10.1016/j.bbagen.2013.06.006" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g165-antichondrsulf.REF.2">Kitagawa H. Using sugar remodeling to study chondroitin sulfate function. <span><span class="ref-journal">Biol Pharm Bull. </span>2014;<span class="ref-vol">37</span>(11):1705–12.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/25366475" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 25366475</span></a>] [<a href="http://dx.crossref.org/10.1248/bpb.b14-00613" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g165-antichondrsulf.REF.3">Mikami T, Kitagawa H. Sulfated glycosaminoglycans: their distinct roles in stem cell biology. <span><span class="ref-journal">Glycoconj J. </span>2017 Dec;<span class="ref-vol">34</span>(6):725–735.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/27709407" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 27709407</span></a>] [<a href="http://dx.crossref.org/10.1007/s10719-016-9732-9" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593826_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g165-antichondrsulf.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="g165-antichondrsulf.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593826/bin/g165-antichondrsulf-Image001.jpg" alt="Figure 1: . Immunocytochemistry of Saos-2 cells, a human osteosarcoma cell line, using an anti-CS antibody CS-56 (diluted 1: 200)." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Immunocytochemistry of Saos-2 cells, a human osteosarcoma cell line, using an anti-CS antibody CS-56 (diluted 1: 200). The CS-56 immunoreactivity was eliminated by pretreatment with chondroitinase ABC (ChABC(+)). Cell nuclei were counterstained with DAPI.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g165-antichondrsulf.F2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593826/bin/g165-antichondrsulf-Image002.jpg" alt="Figure 2: . Immunohistochemistry of tibialis anterior (TA) muscles of wild-type mice using an anti-CS antibody 2B6." /></div><h3><span class="label">Figure 2: </span></h3><div class="caption"><p>Immunohistochemistry of tibialis anterior (TA) muscles of wild-type mice using an anti-CS antibody 2B6. TA muscle sections pretreated with chondroitinase ABC (ChABC(+)) were labeled with 2B6 (diluted 1: 200) and an anti-laminin antibody (rabbit IgG, diluted 1: 1,000, Sigma), followed by nuclear staining with DAPI. 2B6 immunoreactivity was detected preferentially around individual myofibers, especially laminin-positive areas that correspond to basal lamina and endomysium.</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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