Ultravision dab plus substrate detection system

Formalin-fixed-paraffin-embedded (FFPE) brain tumor sections were deparaffinized at 60°, rehydrated through triple washes with Xylene, 100% EtOH, 95% EtOH, 70% EtOH, 50% EtOH, and ddH2O. After heat-mediated antigen retrieval in 5% citrate-buffer, 3% hydrogen peroxidase was used to quench endogenous peroxidase prior to blocking with 3% bovine serum albumin (BSA) and 1% horse serum (HS). Following blocking, the tumor sections were incubated with primary antibodies overnight at 4° or 2 h at room temperature. The sections were then incubated with horseradish peroxidase (HRP)-conjugated polymer (Biocare Medical, Concord, CA) for 45 min and then with diaminobenzidine using the Ultravision DAB Plus Substrate Detection System (Thermo Fischer Scientific, Waltham, MA) for 5 min at room temperature, followed by hematoxylin staining for 1 min. The tumor sections were then washed, dehydrated, and mounted with coverslips. The light microscopy images were taken with Leica DFC295 Bright Field microscope.