Step Protocol Bake, Dewax, rehydrate Leica Bondmaxautostainer standard bake at 60°C for 30min, followed by standard dewax and rehydrate program using Leica Dewax solution (Cat no. AR9222), 100% ethanol and deionized water. Antigen retrieval Leica ER2 buffer pH9 20 min 100°C Wash Bondmax standard wash protocol with Bond wash solution (Cat no. AR9590) Block endogenous peroxidase Peroxidase block at 23°C for 5 min with Bond Polymer Refine kit (Cat no. DS9800) Wash Bondmax standard wash protocol with Bond wash solution Primary antibody Diluted 1:50 in Bond antibody diluent (Cat no. AR9352) for 1 hr at 23°C Wash Bondmax standard wash protocol with Bond wash solution Detection of bound antibody Post Primary Antibody (8 min at 23°C) followed by standard wash and polymer from Bond Polymer Refine kit (Cat no. DS9800) for 8 min at 23°C Wash Bondmax standard wash protocol with Bond wash solution Color development DAB 10min at 23°C from Bond Polymer Refine kit (Cat no. DS9800) Wash Standard Bond wash with distilled water. Counterstain Haematoxylin 5 min from Bond Polymer Refine kit (Cat no. DS9800) Wash Standard Bond wash with distilled water. Mount Aquatex aqueous mountant (Cat no 3631235, VWR).

Table 2: Automated IGF-1R IHC protocol. This protocol was tested on a Leica Bondmax autostainer to evaluate antibodies #9750 and #14534. The method produced acceptable specificity and sensitivity in cell controls (Figure 1D) but generated very weak staining in FFPE tissue (see Figure 2A) and is not recommended for future studies.