Immunohistochemical Analysis of FFPE Cells

The general procedure has been described previously [30 (link)]. In short, immunocytochemistry was performed on freshly cut 3μm thick slides from FFPE cell pellets on the automated IHC staining system Discovery XT (Roche/Ventana, Tuscon, Arizona, USA). The following antibodies were used: rabbit-anti-CPE diluted 1:500 (Novus Biologicals); rabbit-anti-GLUT1 diluted 1:200 (Abcam), rabbit-anti-LDHA diluted 1:100 (C4B5) and rabbit-anti-P-AMPKα (T172) diluted 1:100 (40H9) (all from Cell Signaling). The staining procedure on the Discovery XT contained heat treatment of the slides (95° and 100°Celsius), CC1 cell conditioning and incubation with primary antibodies for 32 minutes. As secondary antibodies we used OMap anti-Rb HRP (Multimer HRP) for 16 minutes. As substrate we used diaminobenzidine (DAB) CM followed by a drop of H₂O₂. Copper was added for signal enhancement as Copper CM for 4 minutes. Slides were counterstained with hematoxylin and mounted.