Xylene

Slides were deparaffinized in xylene (Kanto Chemical, Pulau Pinang, Malaysia) (two times) for 10 min. Endogenous peroxidase was inactivated by incubation with 0.3% hydrogen peroxide (Wako) in methanol (Kanto Chemical) for 30 min at room temperature. The tissue was digested with 20 μg/ml proteinase K solution (Life Technologies) in PBS for 15 min at room temperature. The slides were then denatured with 50% formamide (Kanto Chemical) in 4× sodium saline citrate (SSC; Takara Clontech, Kyoto, Japan) at 85°C for 10 min. The Y chromosome probe was denatured with 50% formamide (Kanto Chemical) in 4× SSC for 5 min at 75°C, added to the denatured tissue, covered with Parafilm ® (Pechiney Plastic Packaging, Menasha, WI, USA), and incubated in a humid chamber overnight at 37°C. The Parafilm ® were then removed in 4× SSC, and the slides were washed in 50% formamide (Kanto Chemical) in 2× SSC (Takara Clontech) for 1 h at 37°C and then in 0.1× SSC (Takara Clontech) for 2 h at 37°C. The slides were incubated with anti-digoxigenin-POD (Roche Diagnostics) and visualized using the TSA biotin system (PerkinElmer, Akron, OH, USA) and ImmPACT TM DAB peroxidase substrate (Vector Laboratories, Burlingame, CA, USA), following the manufacturer's guidelines. The sections were counterstained with hematoxylin (Muto Pure Chemicals).