Chondroitin sulfates are unbranched sulfated polysaccharides composed of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid. Due to differential sulfation of hydroxyl groups and C5 epimerization of glucuronic acid, chondroitin sulfate shows highly diverse structures depending on the developmental stage, aging, pathological states and tissue sources (Fig. 1). There are several monoclonal antibodies that recognize various chondroitin sulfate epitopes. Immunohistochemical staining using these antibodies reveals the localization of chondroitin sulfate epitopes in the tissue sections. |
| Category | Glycosaminoglycans |
| Protocol Name | Immunohistochemical localization of chondroitin sulfate |
Authors
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Maeda, Nobuaki
Department of Brain Development and Neural Regeneration, Tokyo Metropolitan Institute of Medical Science
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Reagents
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Chondroitinase ABC protease free (Sigma Aldrich, St. Louis, MO) |
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MO-225 (mouse IgM, Seikagaku Corp., Tokyo, Japan) |
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CS-56 (mouse IgM, Sigma-Aldrich) |
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VECTASTAIN ABC mouse IgM Kit (Vector Laboratories Inc., Burlingame, CA) |
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Permount (Thermo Fisher Scientific Inc., Waltham, MA) |
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APS-coated slide glass (Matsunami Glass Inc., Ltd., Osaka, Japan) |
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Instruments
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Preparation of tissue sections |
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Perfuse the animals with 4% paraformaldehyde/ 0.1 M sodium phosphate buffer, pH 7.4, via the left ventricle, and wash out the solutions from right atrium. |
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Postfix the perfused tissues in 4% paraformaldehyde/ 0.1 M sodium phosphate buffer, pH 7.4, overnight at 4°C. |
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Embed the tissues in paraffin after dehydration through a graded alcohol series. |
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Cut the 5-μm-thick tissue sections using a rotary microtome. |
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Mount the sections onto APS-coated slide glass. |
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Store sections at room temperature. |
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Deparaffinize the sections with xylene and rehydrate them in graded alcohol series. |
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Incubate the sections in 2.5% H2O2/ PBS for 30 min at room temperature to inactivate endogenous peroxidase. |
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Wash the slide with PBS three times. |
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Incubate the sections in 2% BSA/ 4% goat serum/ PBS for 30 min at room temperature. |
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Wash the slide with PBS three times. |
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Incubate a part of the sections in 50 mU/mL chondroitinase ABC/ 100 mM Tris-HCl, pH 8.0/ 30 mM sodium acetate for 60 min at 37°C. The chondroitinase-treated sections serve as negative controls. |
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Wash the slide with PBS three times. |
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Incubate the sections in MO-225 or CS-56 diluted 1:200 in 0.1% BSA/ PBS for 60 min at room temperature. |
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Wash the slide with PBS three times. |
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Incubate the sections in biotinylated anti-mouse IgM diluted 1:200 in 0.1% BSA/ PBS for 60 min at room temperature. |
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Wash the slide with PBS three times. |
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Incubate the sections in the ABC solution in 0.1% BSA/ PBS for 60 min at room temperature. |
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Wash the slide with PBS three times. |
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Incubate the sections in 0.1% diaminobenzidine/ 0.02% H2O2/ PBS. |
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Wash the slide with distilled water three times. |
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Dehydrate sections with graded alcohol series and xylene. |
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Mount the sections in Permount. |
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| Discussion | Fig. 2. shows the immunohistochemical staining of the sagittal brain sections from postnatal day 7 mouse with CS-56 and MO-225 monoclonal antibodies. CS-56 epitopes are widely observed in the brain including cerebral cortex (Cx) and cerebellum (Ce). In contrast, the staining of MO-225 is very weak in the cerebral cortex, although cerebellum is heavily stained by this antibody. MO-225 recognizes specifically the D unit-containing sequences (Yamagata et al., 1987), whereas CS-56 reacts with various chondroitin sulfate preparations including chondroitin sulfate A, C and D (Fig. 1). These observations suggest that chondroitin sulfate is widely distributed in the brain but the expression of D unit is downregulated selectively in the cerebrum. This was confirmed by the disaccharide composition analyses of chondroitin sulfate from cerebrum and cerebellum (Ishii & Maeda, 2008a and b). |
| Figure & Legends |
Figure & Legends 
Fig. 1. Structures of disaccharide units of chondroitin sulfate
Fig. 2. Immunohistochemical staining of the mouse brain with CS-56 and MO-225 |
| Copyrights |
 Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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| Date of registration：2015-08-18 14:25:52 |
- Yamagata, M., Kimata, K., Oike, Y., Tani, K., Maeda, N., Yoshida, K., Shimomura, Y., Yoneda, M., AND Suzuki S. (1987) A monoclonal antibody that specifically recognizes a glucuronic acid 2-sulfate-containing determinant in intact chondroitin sulfate chain. J. Biol. Chem. 262, 4146–4152 [PMID : 2435733]
- Maeda, N., He, J., Yajima, Y., Mikami, T., Sugahara, K., AND Yabe, T. (2003) Heterogeneity of the chondroitin sulfate portion of phosphacan/6B4 proteoglycan regulates its binding affinity for pleiotrophin/heparin binding growth-associated molecule. J. Biol. Chem. 278, 35805–35811 [PMID : 12840014]
- Ishii, M., and Maeda, N. (2008a) Spatiotemporal expression of chondroitin sulfate sulfotransferases in the postnatal mouse cerebellum. Glycobiology 18, 602–614 [PMID : 18480156]
- Ishii, M., and Maeda, N. (2008b) Oversulfated chondroitin sulfate plays critical roles in the neuronal migration in the cerebral cortex. J. Biol. Chem. 283, 32610–32620 [PMID : 18819920]
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