Antigen retrieval solution ph 9

Each excised xenograft tumor was fixed by formaldehyde, embedded in paraffin, and then sliced on glass slides. For immunohistochemistry, paraffin-embedded tumor section was deparaffinized, rehydrated, stained with hematoxylin (Dako) and eosin (Sakura Finetek), and analyzed by IHC using antibodies as described above. Briefly, the tumor sections were treated with xylene and ethanol, and incubated with Antigen Retrieval Solution pH9 (Nichirei) to retrieve antigens. Then, tumor sections were incubated with 3% H₂O₂ solution (Wako) for peroxidase blocking and Serum-free Protein Blocking Reagent (Dako), according to manufacturer's instructions. These sections were incubated with primary antibodies diluted in the Antibody Diluent Solution (Dako), followed by incubation with Histofine Simple Stain MAX PO (Nichirei), and then stained with substrate-chromogen (Histofine DAB kit, Nichirei). Finally, tumor sections were counterstained with hematoxylin (Dako) and observed in a bright-field microscope (Leica).