Benchmark ultra ihc system

Histology and IHC were performed as previously described (Liu et al., 2015 (link)). Briefly, xenograft tissues were collected, fixed in 4% buffered formalin-saline at room temperature for 24 h, embedded in paraffin blocks, and then sections of 4 µM thickness were mounted on glass slides. For IHC assays, slides were immersed in 3% H₂O₂ for 5 min, washed, incubated for 15 min in 5% BSA to block non-specific binding sites and finally incubated in primary antibodies overnight at 4°C. The slides were washed the next day and then incubated with biotinylated anti-rabbit secondary antibody using a DAB kit (Dako) according to the manufacturer's protocol. Slides were counterstained with Hematoxylin. An anti-WT1 antibody that was raised against a peptide mapping at the C-terminal region of WT1 (C-19, Santa Cruz Biotechnology) was used. EGFR and phospho-EGFR IHC were conducted with anti-EGFR (D38B1) and anti-phospho-EGFR (Y1068) antibodies, respectively, obtained from Cell Signaling Technology. IHC staining of patient samples was performed with the VENTANA BenchMark ULTRA IHC system. ERBB2 IHC was conducted using an anti-ERBB2 antibody (4B5) purchased from Roche.

In silico deconvolution results regarding B cells were validated by anti-CD20 immunohistochemistry analyses. Double-blinded labeled microscopic slides were prepared from the FFPE tumor samples, processed in the BenchMark ULTRA IHC System, incubated with CONFIRM anti-CD20 L26 primary antibody (Ventana Medical Systems, Oro Valley AZ, USA) and ultraView Universal DAB Detection Kit as a secondary antibody (Ventana Medical Systems, Oro Valley AZ, USA).
Following whole slide scanning on the Aperio LV1 System (Leica Biosystems, Richmond IL, USA), tumor and normal areas were defined by two pathologists. The slides were then quantified by two separate methods: (1) An independent intratumoral and peritumoral CD20 ${}^{+}$ percentage estimation by two experienced pathologists, with the joint assessment of a third senior pathologist in cases of discrepancy; (2) Independent digital image analysis and quantification using immunohistochemistry profiler software [44 (link)]. Digitally analyzed tumor, normal, and total CD20 ${}^{+}$ percentages were calculated by dividing the CD20 ${}^{+}$ pixel count by the number of total pixels.