Anti rabbit anti mouse envision

The expression of OCT4 and NANOG was immunohistochemically evaluated using the protocol as described previously^{22 (link)–26 (link)}. Briefly, sequential TMA sections were dewaxed at 60 °C for 20 minutes, rehydrated in xylenes, and underwent graded ethanol treatment. Antigen was retrieved by autoclaving tissue sections for 10 minutes in sodium citrate buffer (pH 6.0). Endogenous peroxidase and nonreactive staining were blocked by 3% H₂O₂ for 20 minutes at room temperature. Then, the tissue sections were incubated overnight at 4 °C with the following antibody dilutions: anti-OCT4 antibody (ab18976; Abcam, UK) using a 1:100 dilution, and anti-NANOG antibody (ab109250; Abcam, UK) using a 1:150 dilution. After 3 washes in Tris-buffered saline (TBS), sections were incubated with anti-rabbit/anti-mouse envision (Dako, Denmark), as the secondary antibody, for 15 minutes. TMA slides were treated with 3,3′-diaminobenzidine (DAB, Dako) substrate as a chromogen for 10 minutes at room temperature. Sections were lightly counterstained with hematoxylin, dehydrated in alcohol, cleared with xylenes, and mounted. For negative controls, the primary antibody step was replaced with TBS, and only the secondary antibody was used. Human seminoma tissues were used as a positive control for OCT4 and NANOG staining.