In Situ Hybridization Protocol for Tissue Sections

Tissue sections were fixed with 4% paraformaldehyde for 1 hour, followed by three washes of 0.1M phosphate buffer, air-dried, and stored at −20°C until use. Sections were processed for in situ hybridization as described (Cheng et al., 2002 (link), Zhang et al., 2009 (link)). Briefly, sections were dried at room temperature for 2 hours, followed by pretreatment of proteinase K (1 μg/ml). Sections were air-dried and then hybridized with riboprobes by incubation at 60°C for 18 hours. After hybridization, tissue was treated to RNAase (20 μg/ml) (Sigma-Aldrich, St. Louis, MO), decreasing salinity washes and a 30 minute high stringency (68°C) wash. After dehydration and air-drying, tissue sections were exposed to Kodak Biomax film for 5–15 days. Specific hybridization signals were quantitatively analyzed using a videobased computer image analysis system (MCID, Imaging Research, Ontario, Canada). A calibration curve of optical density versus radioactivity (dpm/mg tissue wet weight) was constructed using ¹⁴C-standards. Figures were prepared by using Adobe Photoshop (Adobe Systems Inc.).