Procedure for in Situ Hybridization of DNA

Paraffin sections (6 p,m thick) of formalin-fixed tissues are mounted onto silanized glass slides and air-dried at 60°C (Henke and Ayhan, 1994). They are deparaffinized, rehydrated, and air-dried. The slides are placed in aplastic Coplinjar filled with lOmM sodium citrate buffer (pH 6.0) and microwaved (720 W) for 1 min this time is measured after reaching the boiling point. After treating with 1 M sodium thiocyanate for 10 min at 80°C, the sections are washed with distilled water and then digested with pepsin (3mg/ml in 0.2 N HC1) for 6 min at 37°C. Both the heating and the pepsin digestion durations must be 

Each section is covered with 100 xl of freshly prepared hybridization solution Deionized formamide (65%) 

Sections are covered with cover-slips, sealed with rubber cement, denatured by heating at 78°C in a water bath for 10 min, and hybridized overnight at 37°C. The coverslips are carefully removed by floating the slides in 2 X SSC, and the sections are washed twice for 10 min each in a mixture of 2 X SSC and 50% formamide at 40°C, and then three times in PBS. Endogenous peroxidase is blocked by incubation in 1% H202 for 15 min. 

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