St5020

TMA was constructed at the Stephenson Cancer Center's tissue pathology core using Veridiam VTA-100 Tissue Arrayer [7 (link)]. IHC-staining of the TMA with the antibodies of interest was carried out at the SCC tissue pathology core that utilizes automated Leica Bond III for IHC staining. In brief, FFPE tissues were sectioned at desired thickness (4µm) and mounted on positively charged slides. The slides were dried overnight at room temperature and incubated at 60°C for 45 minutes followed by deparaffinization and rehydration in an automated Multistainer (Leica ST5020). Subsequently, these slides were transferred to the Leica Bond-III^TM, treated for target retrieval at 100°C for 20 minutes in a retrieval solution, either at pH 6.0 or pH 9.0. Endogenous peroxidase was blocked using peroxidase-blocking reagent, followed by the selected primary antibody incubation for 60 minutes. For the secondary antibody, post-primary IgG-linker and/or Poly-HRP IgG reagents was used. 3, 3′-diaminobenzidine tetrahydrochloride (DAB) was used as the chromogen and the slides were counterstained with hematoxylin. Completed slides were dehydrated (Leica ST5020), and mounted (Leica MM24). Antibody specific positive control and negative control (omission of primary antibody) were parallel-stained.

Immunohistochemistry was performed according to manufacturer's protocol using Leica Bond™ Polymer Refine Detection system (DS 9800). In brief, formalin-fixed paraffin-embedded tissues were sectioned (4 μm) and mounted on positively charged slides. The slides were dried overnight at room temperature and incubated at 60°C for 45 minutes followed by deparaffinization and rehydration in an automated Multistainer (Leica ST5020). Subsequently, these slides were transferred to the Leica Bond-III™, treated for target retrieval at 100°C for 20 minutes with a retrieval buffer, pH 6.0. Endogenous peroxidase was blocked using peroxidase-blocking reagent, followed by the primary antibody, SPAG9N (Aviva OAAB04262; 1:50 dilution) incubation. For the secondary antibody, post-primary IgG-linker and/or Poly-AP IgG reagents were used. The substrate chromogen, 3, 3′-diaminobenzidine tetrahydrochloride (DAB) detects the complex as brown precipitate, while hematoxylin counterstain the cell nuclei (blue). The slides were then dehydrated (Leica ST5020), and mounted (Leica MM24). Antibody specific positive and negative (omission of primary antibody) controls were parallel stained.