In Situ Hybridization Tissue Imaging

Procedures for In situ hybridization were carried out similarly as described [6 (link), 7 (link)]. Tissue sections were cut at −20 °C, and then fixed with 4 % paraformaldehyde, followed by three washes of 0.1 M phosphate buffer, air-dried, and stored at −20 °C until use. For In situ hybridization, sections were dried at room temperature, followed by pretreatment of proteinase K (1 μg/ml). Sections were then air-dried and hybridized with S [35 (link)]-labelled riboprobes by incubation at 60 °C for 18 h. After hybridization, tissue sections were treated with RNAase (20 μg/ml) (Sigma-Aldrich, St. Louis, MO), decreasing salinity washes and high stringency (68 °C) wash. After dehydration and air-drying, tissue sections were exposed to Kodak Biomax film. Images were captured with image analysis system (MCID, Imaging Research, Ontario, Canada).