Most bones are formed from the cartilage primordium, which process is termed endochondral ossification. Therefore, impairment of cartilage development caused skeletal defects, including dwarfism. Cartilage contains a large amount of proteoglycans (PGs). Genetic mutations that affect biosynthesis of proteoglycan core proteins and glycosaminoglycans often impair cartilage development, causing skeletal defects. Examples are cartilage matrix deficiency, a natural knockout mouse of aggrecan core protein, perlecan-null mice and their human counterpart Schwartz-Jampel syndrome. In addition, knockout mice of PAPS synthase-2, brachymorphic, bm/bm, and nucleotide-sugar transporter SLC35D1, exhibit skeletal defects. As mutations of genes involving PGs and glycosaminoglycans may cause skeletal defects, analysis of skeleton is an initial step of the mutant mice. This section describes both gross and histological analysis of mice skeleton. |
| Category | Nucleotide sugar transporters |
| Protocol Name | Skeletal analysis of mice |
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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alcian blue 8GX (Sigma-Aldrich, St. Louis, MO), preparation: 80 mL ethanol, 20 mL acetic acid, 15 mg alcian blue |
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alizarin red S (Chroma, Kongen, Germany), 0.001% alizarin red S in 1% KOH |
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K-CX decalcifying reagent (Falma Co., Ltd., Tokyo, Japan) |
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Anti-collagen II antibody, rabbit polyclonal (Research Diagnostics, Inc., Flanders, NJ) |
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Anti-aggrecan antibody (12/21/1-C-6, Developmental Studies Hybridoma Bank, Iowa City, IA) |
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Anti-link protein antibody (9/30/8-A-4, Developmental Studies Hybridoma Bank) |
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Bovine testicular hyaluronidase (Sigma-Aldrich) |
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Chondroitinase ABC (Seikagaku Corp., Tokyo, Japan) |
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HistomouseTM kit (Zymed/Life Technologies, Carlsbad, CA) |
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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. |
Double staining with alcian blue and alizarin red for skeleton |
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Remove organs and skin as much as possible. |
Comment 1
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Stain for 2–3 days in alcian blue solution. |
Comment 1
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Dehydrate for five days in 100% ethanol. |
Comment 0
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Immerse the specimen in 1% KOH for 3–5 days. |
Comment 1
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Stain the specimen in alizarin red solution for 2 days. |
Comment 0
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Destain alizarin red by immersing in 1% KOH for 1–2 days. |
Comment 0
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Store the double-stained specimen in 50–100% glycerin. |
Comment 0
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2. |
Immunohistochemical analysis of mouse skeleton |
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Fix the specimen with 10% buffered formalin. |
Comment 0
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Decalcify the specimen with K-CX overnight. |
Comment 1
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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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Pretreat the section slides to retrieve antigen. |
Comment 1
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Wash with PBS for 5 min × three times. |
Comment 0
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Treat with 10% goat serum for 30 min–1 h at room temperature. |
Comment 0
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Treat with a primary antibody for 2 h at room temperature or 4°C overnight in moist chamber. |
Comment 1
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Wash with PBS for 5 min × three times. |
Comment 0
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| 14) |
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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Immunodetect with the Liquid DAB+ substrate chromogen system. |
Comment 0
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| Copyrights |
 Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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| Date of registration：2015-07-06 14:00:25 |
- Peters, P. W. J. (1977) Double staining of fetal skeletons for cartilage and bone. In Methods in prenatal toxicology (Eds. Neuberg, D., Merker, H-J., Kwasigrch, T. E.) 153–154.
- Watanabe, H. and Yamada, Y. (1999) Mice lacking link protein develop dwarfism and craniofacial abnormalities. Nature Genet. 21, 225–229 [PMID : 9988279]
- Arikawa-Hirasawa, E., Watanabe, H., Takami, H., Hassell. J. R., and Yamada, Y. (1999) Perlecan is essential for cartilage and cephalic development. Nature Genet. 23, 354–358 [PMID : 10545953]
- Matsumoto, K., Kamiya, N., Suwan, K., Atsumi, F., Shimizu, K., Shinomura, T., Yamada, Y., Kimata, K., and Watanabe, H. (2006) Identification and characterization of versican/PG-M aggregates in cartilage. J. Biol. Chem. 281, 18257–18263 [PMID : 16648631]
- Sato, T., Kudo, T., Ikehara, Y., Ogawa, H., Hirano, T., Kiyohara, K., Hagiwara, K., Togayachi, A., Ema, M., Takahashi, S., Kimata, K., Watanabe, H., and Narimatsu, H. (2011) Chondroitin sulfate N-acetylgalactosaminyltransferase 1 is necessary for normal endochondral ossification and aggrecan metabolism. J. Biol. Chem. 286, 5803–5812 [PMID : 21148564]
- Hiraoka, S., Furuichi, T., Nishimura, G., Shibata, S., Yanagishita, M., Rimoin, D. L., Superti-Furga, A., Nikkels P. G., Ogawa, M., Katsuyama, K., Toyoda, H., Kinoshita-Toyoda, A., Ishida, N., Isono, K., Sanai, Y., Cohn, D. H., Koseki, H., and Ikegawa, S. (2007) Nucleotide-sugar transporter SLC35D1 is critical to chondroitin sulfate synthesis in cartilage and skeletal development in mouse and human. Nature Med. 13, 1363–1367 [PMID : 17952091]
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