Diaminobenzidine substrate chromogen solution dab

Formalin fixed, paraffin embedded lung tissues were cut into 5 μm thick sections, stained with hematoxylin/eosin and evaluated by light microscopy. Immunohistochemical staining was performed to detect EGFP expressed in tumor cells. Slides were deparaffinized and rehydrated in phosphate buffered saline solution (10 mM, pH 7.2). The tissue epitopes were demasked using the automated water bath heating process in Dako PT Link (Dako, Glostrup, Denmark); the slides were incubated in pH 6.0 citrate retrieval buffer at 98°C for 20 minutes. The slides were subsequently incubated for 60 minutes at room temperature with the primary rabbit polyclonal antibody against GFP (Abcam, anti-GFP, ab290) diluted 1:500 in Dako REAL antibody diluent (Dako, Glostrup, Denmark) and immunostained using anti-mouse/anti-rabbit immuno-peroxidase polymer (EnVision FLEX/HRP, Dako, Glostrup, Denmark) for 30 minutes at room temperature, according to the manufacturer’s instructions. For visualization, the slides were reacted with diaminobenzidine substrate-chromogen solution (DAB, Dako, Glostrup, Denmark) for 5 minutes. Finally, the slides were counterstained with hematoxylin.

Slides were deparaffinized and rehydrated in phosphate buffered saline solution (10 mM, pH 7.2). The tissue epitopes were demasked using the automated water bath heating process in Dako PT Link (Dako, Glostrup, Denmark); the slides were incubated in TRIS-EDTA retrieval solution (10 mM TRIS, 1 mM EDTA pH 9.0) at 98 °C for 20 min. The slides were subsequently incubated for 1 h at room temperature with the primary mouse monoclonal antibody against Beta-Catenin (Dako, β-Catenin-1, IR702, Ready-to-Use) and immunostained using anti-mouse/anti-rabbit immuno-peroxidase polymer (EnVision FLEX/HRP, Dako, Glostrup, Denmark) for 30 min at room temperature, according to the manufacturer’s instructions. For visualization, the slides were reacted with diaminobenzidine substrate-chromogen solution (DAB, Dako, Glostrup, Denmark) for 5 min. Finally, the slides were counterstained with haematoxylin. Βcatenin positivity of epithelial cells in the colon was used as a positive control, same tissue with omitting of the primary antibody served as negative control.

Slides were deparaffinised, rehydrated and immersed in phosphate buffered saline solution (10 mM, pH 7.2). Tissue epitopes were demasked through revitalisation in TRIS-EDTA retrieval solution (10 mM TRIS, 1 mM EDTA, pH 9,0) at 98 °C for 20 min in Dako PT Link (Dako, Glostrup, Denmark). The slides were subsequently incubated for 1 h at room temperature with primary mouse monoclonal antibody against β-catenin (IR702, Ready-to-Use, Dako) and immunostained using anti-mouse/anti-rabbit secondary antibody (EnVision FLEX/HRP, Dako) for 30 min at room temperature. The reaction was visualised by diaminobenzidine substrate-chromogen solution (DAB, Dako) which was applied for 5 min. Ultimately, the slides were counterstained with hematoxylin. Non-neoplastic testicular tissue was used as a positive control and the same tissue without incubation in primary antibody represented the negative control. Representative images were captured with Olympus BX40 microscope (Olympus Corporation, Tokyo, Japan) and Canon EOS 1000D (Canon Inc., Tokyo, Japan).