Keratan sulfate (KS) is mainly present in cornea, cartilage and brain, exhibiting various in vivo functions. To date, a number of anti-KS antibodies have been used for KS studies. This protocol introduces some anti-KS antibodies and shows methods of immunostaining for KS. |
| Category | Glycosaminoglycans |
| Protocol Name | Application of anti-GAG antibody and biotinylated hyaluronan binding protein(bHABP) [3] ~ Immunostaining of keratan sulfate |
Authors
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Watanabe, Hideto
Institute for Molecular Science of Medicine, Aichi Medical University
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| KeyWords |
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Reagents
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anti-KS antibody
Name (Distribuors) Antigen
5D4 (Seikagaku Corp., Tokyo, Japan) Cartilage proteoglycan digested with chondroitinase ABC
EFG-11(Varieous companies) Cartilage proteoglycan digested with chondroitinase ABC
3H1 (DSHB) Brain KS on phosphacan
I22 (DSHB) New Zealand white rabbit cornea
MZ15 (DSHB) Porcine larynx
*DSHB; Developmental Studies Hybridoma Bank
http://dshb.biology.uiowa.edu/ |
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Antibody diluent with background reducing components (DAKO, Glostrup, Denmark, S3022) |
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LSABTM2 biotinylated link for streptavidin HRP/AP (DAKO, K1015) |
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LSABTM2 streptavidin-HRP (DAKO, K1016) |
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Liquid DAB+ substrate chromogen system (DAKO, K3468) |
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10% goat serum (Nichirei Corp., Tokyo, Japan) |
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| Methods |
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1. |
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| 1) |
Fix the specimen with 4% paraformaldehyde, 10% buffered formalin. |
Comment 1
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| 3) |
Prepare histology section slides at an appropriate thickness. |
Comment 1
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Deparaffinize the section slides. |
Comment 0
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Treat the section slides with 0.3–3% H2O2 in methanol for 10 min. |
Comment 0
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| 7) |
Treat with 10% goat serum for 30 min–1 h at room temperature. |
Comment 0
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| 9) |
Treat with an anti-KS antibody diluted with an antibody diluent (For 5D4, 50–200× dilution) for 2 h at room temperature or 4°C overnight in moist chamber. |
Comment 1
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| 10) |
Wash with PBS for 5 min × three times. |
Comment 0
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| 11) |
Treat with biotinylated link and incubate for 20 min or longer at room temperature. |
Comment 0
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Wash with PBS for 5 min × three times. |
Comment 0
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Treat with streptavidin HRP and incubate for 15 min or longer at room temperature. |
Comment 0
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Wash with PBS for 5 min × three times. |
Comment 0
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| 15) |
Immunodetect with the Liquid DAB+ substrate chromogen system. |
Comment 0
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| Notes |
- The immunostaining method above is an example. Researchers could utilize their own methods.
- Seikagaku Corp. discontinued supplying 5D4. Other providers/distributors should be found.
- Studies using a novel monoclonal anti-KS antibody, BKS-1, have been published. BKS-1 recognizes keratanase-digested KS-stub.
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| Copyrights |
 Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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| Date of registration：2017-01-23 15:52:52 |
- Caterson, B., Christner, J. E., and Baker, J. R. (1983) Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate: monoclonal antibodies to cartilage proteoglycan. J. Biol. Chem. 258, 8848–8854 [PMID : 6223038]
- Zhang, H., Muramatsu, T., Murase, A., Yuasa, S., Uchimura, K., and Kadomatsu, K. (2006) N-acetylglucosamine 6-O-sulfotransferase-1 is required for brain keratan sulfate biosynthesis and glial scar formation after brain injury. Glycobiology 16, 702–710 [PMID : 16624895]
- Akhtar, S., Kerr, B. C., Hayes, A. J., Hughes, C. F., Meek, K. M., and Caterson, B. (2008) Immunohistochemical localization of keratan sulphate proteoglycans in cornea, sclera, and limbus using a keratanase-generated neoepitope monoclonal antibody. Invest. J. Ophthalmol. 49, 2424–2431 [PMID : 18515583]
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