R t u vectastain detection kit

Tumor and metastasis samples obtained from animals were embedded in paraffin wax and cut in 10 µm sections for hematoxylin–eosin staining. For lung immunohistochemistry, 5 μm sections were cut, dewaxed, and rehydrated. Sections were treated with absolute methanol/3% H₂O₂ for 15 min to quench endogenous pseudoperoxidase activity, rinsed in distilled water and then in Tris–HCl buffer. After blocking non-specific binding for 20 min with horse serum (Vecton, Carpinteria, CA, USA), tissue sections were incubated with anti-S-Nitroso-Cysteine. The antibody was reconstituted and diluted in 0.01 M PBS containing 1% IgG-free bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) and 0.02% NaN₃. Bound antibodies were detected using the R.T.U VECTASTAIN Detection Kit (Vector Laboratories) and peroxidase was visualized with Liquid-3,3ʹdiaminobenzidine (DAB) + Substrate Chromogen System (DAKO). Slides were counterstained with hematoxylin–eosin and mounted with mounting medium (DAKO). Cells were visualized using a light microscope (Zeiss).

Tumor samples were fixed with Bouin, embedded in paraffin wax, cut into 5 μm sections and adhered to poly-L-lysine-coated slides. Tissue sections were dewaxed and rehydrated. Sections were treated with absolute methanol/3% H₂O₂ for 5 min to quench endogenous pseudoperoxidase activity, rinsed in distilled water and then in Tris–HCl buffer. After blocking non-specific binding for 15 min with horse serum (Vecton, Carpinteria, CA, United States), tissue sections were incubated with anti-S-Nitroso-Cysteine. The antibody was reconstituted and diluted in 0.01 M PBS containing 1% IgG-free bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, United States) and 0.2% NaN3. Bound antibodies were detected using the R.T.U VECTASTAIN Detection Kit (Vector Laboratories) and peroxidase was visualized with Liquid-3,3′diaminobenzidine (DAB) + Substrate Chromogen System (DAKO). Controls included omission of primary antibody. Slides were counterstained with hematoxylin-eosin and mounted with mounting medium (DAKO). Cells were visualized using a light microscope (Zeiss).