Optiview universal dab detection kit

Immunohistochemistry (IHC) for monoamine oxidase A (MAO-A) was performed on FFPE whole sections of all the analyzed primary tumors and mesenteric metastases. Sequential 4 µm thick FFPE sections were stained for MAO-A (EPR7101, ab126751, 1:3200, Abcam) by automated IHC using the Ventana Benchmark ULTRA (Ventana Medical Systems Inc.). In brief, following deparaffinization and heat-induced antigen retrieval with CC1 (no. 950-500, Ventana) for 64 min the tissue samples were incubated with the antibody of interest for 32 min at 37°C. The staining was developed using Optiview universal DAB detection Kit (no. 760-700, Ventana), followed by hematoxylin II counterstain for 8 min and then a blue coloring reagent for 8 min according to the manufacturer’s instructions (Ventana). Positive controls were used on every slide.
Immunohistochemically stained samples were digitalized and four fields of view were representatively selected. Each field of view was exported as an image file on a 10× magnification scale and contained both tumor cells and stroma. The exported images were analyzed using the CellProfiler software (version 3.0, www.cellprofiler.org) (14 (link)). Each image was manually segmented into the tumor and stromal compartments and the average intensity of DAB staining of the segmented area (I/A) was noted.

IHC analysis was conducted with the PD‐L1 IHC 22C3 pharmDx and the Ventana PD‐L1 (SP263) assays on the DAKO Autostainer Link 48 and Ventana BenchMark platforms, respectively. Consecutive 4 μm thick sections cut from the same core specimen were pretreated and stained with the PD‐L1 antibody 22C3 mouse monoclonal primary antibody from pharmDx on a Dako Autostainer Link 48 with EnVision DAB Detection System (Agilent/Dako, Santa Clara, CA, USA) with negative control reagents and cell line run controls, as described in the PD‐L1 IHC 22C3 pharmDx, and PD‐L1 antibody SP263 rabbit monoclonal primary antibody from Ventana on a Ventana Benchmark Ultra with OptiView Universal DAB Detection Kit (Ventana Medical Systems, Tucson, AZ, USA) with a matched rabbit immunoglobulin G–negative control, in accordance with the manufacturers’ instructions, respectively. The detection and quantification of the percentage of immunoreactive tumor cells was performed according to the manufacturers’ recommendations. Briefly, neoplastic cells were considered positive when any cell membrane staining (partial or complete) was present, ignoring pure cytoplasmic immunoreaction. Staining on immune cells was also disregarded. Quantification of immunoreactive neoplastic cells was obtained by evaluating the ratio between stained carcinoma cells and all viable carcinoma cells.

When available, formalin fixed paraffin-embedded tumor samples collected from patients with well-differentiated and dedifferentiated LPS during a previous biopsy or surgery were reviewed by a designated pathologist and analyzed for PD-L1 expression. Immunohistochemical staining for PD-L1 was performed with the Ventana SP263 assay (rabbit monoclonal primary anti–PD-L1 antibody, Ventana Medical Systems, Tucson, AZ), Food and Drug Administration (FDA)-approved complementary diagnostics [11 (link)]), on the Benchmark XT staining systems and Ultra with the OptiView Universal DAB Detection Kit (Ventana Medical Systems), according to the manufacturer’s instructions. Whole tumor section was stained in each case to adequately reflect heterogeneity of expression. Positive PD-L1 expression was defined as staining in ≥ 1% of tumor cells.