Ventana benchmark xt system

IHC staining was performed on the TMA slides using a PD-L1 clone (22C3; Dako, Carpentaria, CA, USA) and stained with Dako Autostainer (Dako) (Figure 1). IHC was also performed with an additional PD-L1 antibody (E1L3N; 1:50, Cell Signaling Technology, Danvers, MA, USA) and CD8 (1:100, Dako, Glostrup, Denmark) on the Ventana Benchmark XT system (Ventana, Tucson, AZ, USA), and visualized with a DAB detection kit (Ventana).
CD8+ TILs were evaluated in at least 3-4 representative high-power field (HPF) areas from each sample, and the mean CD8+ TILs count was scored using the following four-tier scoring system: no lymphocytes (0), 1-10/HPF (1), 11-50/HPF (2), >50/HPF (3) 23 .
We evaluated the PD-L1 expression in TCs or TILs separately, and then in both TC and /or TIL. The PD-L1 positivity was categorized based on the melanoma scoring system (MEL) as reported by Daud et al. 21 (link). The membranous expression in PD-L1 was graded on a five-point scale as follows: 0, no membranous staining; 1, >0%-<1%; 2, ≥1%-<10%; 3, ≥10%-<33%; 4, ≥33%-<66%; 5, ≥66%.

All hematoxylin and eosin-stained tumor-containing ovarian tissue slides were independently reviewed. The chemotherapy response score (CRS) was evaluated according to a previous study [25 (link)]. Briefly, CRS 1 indicated no or minimal tumor response, CRS 2 indicated appreciable tumor response amid a viable tumor that is readily identifiable, and CRS 3 indicated complete or near-complete response with no residual tumor or nodules up to a maximum size of 2 mm.
Conventional IHC analysis was performed on whole sections of FFPE tumor tissue blocks. Four-μm-thick sections of surgically resected tissues were immunostained using a Ventana BenchMark XT system automated stainer (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s recommendations. The sections were incubated with antibodies against RAD51 (clone 14B4; GeneTex, Alton Pkwy Irvine, CA, USA), geminin (clone 10802-1-AP; ProteinTech Group, Chicago, IL, USA), and γH2AX (clone JBW301, Sigma-Aldrich, Dorset, UK). The detailed protocols are summarized in Table S1. After chromogenic visualization using an UltraView Universal DAB Detection Kit (Ventana Medical Systems), the slices were counterstained with hematoxylin, dehydrated in graded alcohols and xylene, and embedded in mounting solution. Appropriate positive and negative controls were concurrently stained to validate the staining method.