Pten clone y184

Immunohistochemistry was performed using tissue arrays of type A and B3 thymomas and thymic carcinomas. 2 μm sections of the tissue arrays were stained on a Ventana Benchmark Ultra (Ventana, Tucson, AZ) with extended heat-induced epitope retrieval with CC1 buffer and the ultraView Universal DAB Detection Kit (Ventana). The following antibodies were employed: ALK (clone 1A4; Zytomed, Berlin, Germany), HER2 (clone 4B5; Ventana), HER3 (clone SP71; Abcam, Milton, UK), MET (clone SP44; Ventana), phospho-mTOR (clone 49F9; Cell Signalling, Danvers, MS), p16^INK4A (clone E6H4; Ventana), PDGFRA (rabbit polyclonal; Thermo Fisher Scientific), PDGFRB (clone 28E1, Cell Signalling), PD-L1 (clone E1L3N; Cell Signalling), PTEN (clone Y184; Abcam) and ROS1 (clone D4D6; Cell Signalling). An immunohistochemial score was determined by multiplying the percentage of positive cells by their respective staining intensity (0 = negative, 1 = weak, 2 = moderate, 3 = strong). Immunohistochemical score (maximum 300) = (% negative x 0) + (% weak x 1) + (% moderate x 2) + (% strong x 3).

A Ventana BenchMark Ultra stainer (Ventana Medical Systems, Tucson, AZ, USA) was used to perform IHC on tissue sections with a thickness of 2 µm. A variety of antibodies were added, including epidermal growth factor receptor (EGFR; clone 3C6; Ventana Medical Systems), HER2 (clone 4B5; Ventana Medical Systems), HER3 (clone SP71; Abcam, Cambridge, UK), mTOR (clone 49F9; Cell Signaling Technology, Danvers, MA, USA), programmed death-ligand 1 (PD-L1; clone E1L3N; Cell Signaling Technology (until mid-2018; as of mid-2018, Nordic Biosite, Stockholm, Sweden, is using the BSR90 clone)), and PTEN (clone Y184; Abcam).
To assess the immunostaining intensity for the antigens EGFR, mTOR, PDGFRA, PDGFRB, and PTEN, a combinative semi-quantitative score was used. For a comprehensive description of the IHC, we refer to our previous work [9 (link)].