Salie Rishard, Li Haitao, Jiang Xi, Rowe David W, Kalajzic Ivo, Susa Mira
Musculoskeletal Disease Area, Oncology Drug Discovery, Novartis Institutes for Biomedical Research, WKL-125.13.18, CH-4002, Basel, Switzerland.
Calcif Tissue Int. 2008 Sep;83(3):212-21. doi: 10.1007/s00223-008-9154-1. Epub 2008 Sep 2.
In situ hybridization (ISH) of adult bone is a difficult task that requires at least 3-5 weeks for decalcification, paraffin embedding, and sectioning. For that reason, bone ISH is often done only on embryonic or newborn animal tissue, leaving unanswered the question of gene expression in adults. Here, we report the development of an ISH system that requires only 7 days for acid-free decalcification, embedding, and sectioning, conditions that are conducive to preservation of tissue mRNA. The tissue cryosections, derived from adult mice 3-12 weeks old, were cut using the CryoJane Tape Transfer system. Paraffin-sectioned and cryosectioned tissue have comparable morphology. Examples are given of cryosections that were hybridized and stained enzymatically with digoxigenin-labeled riboprobes for mRNA found in either bone-forming osteoblasts (type I collagen, osteocalcin, Runx2) or the hypertrophic or proliferating chondrocytes (type X collagen, Runx2).
对成年骨骼进行原位杂交（ISH）是一项艰巨的任务，脱钙、石蜡包埋和切片至少需要3至5周时间。因此，骨骼ISH通常仅在胚胎或新生动物组织上进行，而成年动物基因表达的问题仍未得到解答。在此，我们报告了一种ISH系统的开发，该系统仅需7天即可完成无酸脱钙、包埋和切片，这些条件有利于保存组织mRNA。使用CryoJane胶带转移系统对3至12周龄成年小鼠的组织进行冷冻切片。石蜡切片和冷冻切片的组织形态具有可比性。文中给出了冷冻切片的示例，这些切片用洋地黄毒苷标记的核糖探针进行杂交，并对成骨成骨细胞（I型胶原蛋白、骨钙素、Runx2）或肥大或增殖软骨细胞（X型胶原蛋白、Runx2）中发现的mRNA进行酶促染色。
