A rapid, nonradioactive in situ hybridization technique for use on cryosectioned adult mouse bone

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).