Optiview dab ihc detection kit

Ninety-seven WTS slides were stained using the Dako (Carpinteria, CA, USA.) and Ventana (Tucson, AZ, USA) platforms and their PD-L1 IHC assays. The Dako pharmDx assay was stained with an anti-PD-L1 22C3 mouse monoclonal primary antibody with the EnVision FLEX visualization system on a Dako Autostainer Link 48 system, along with negative control reagents and cell line run controls, as per the manufacturer's instructions [5 (link)]. For the Ventana assay, the sections were stained with an anti-PD-L1 (SP263) rabbit monoclonal primary antibody using the OptiView DAB IHC Detection kit on the BenchMark XT automated staining platform [9 (link)]. For the SP142 assay, the sections were stained with an anti-PD-L1 (SP142) rabbit monoclonal primary antibody using the OptiView DAB IHC Detection kit, followed by the OptiView Amplification Kit, on the BenchMark XT automated platform [6 (link)]. For the E1L3N assay, the sections were stained with an anti-PD-L1 (E1L3N) rabbit XP monoclonal primary antibody (1:100; Cell Signaling Technology, Danvers, MA, USA) with the OptiView DAB IHC Detection kit on the BenchMark XT automated platform.

The immunohistochemistry reaction was performed using BenchMark Ventana Ultra™ (Ventana, Tucson, AZ, USA) platform, through multimer linked to horse radish peroxidase, to detect PD-L1 protein, as previously reported [25 (link)]. The anti-PD-L1 (E1L3N®) XP® Rabbit mAb, Cell Signaling Technology, was used as primary antibody and we used the OptiView DAB IHC Detection Kit, following manufacturer’s guidelines. Placental syncytiotrophoblast was used as positive control tissue. The combined positive score (CPS) was used to measure the expression of PD-L1. CPS corresponds to the ratio between the total of PD-L1 positive cells (tumor cell, lymphocytes and macrophages) and the total of viable tumor cells, multiplied by 100 [26 (link)]. Considering the experience of appropriateness of CPS cutoff in other types of tumors and no agreement of CPS cutoff for CUPs, we used CPS ≥ 1 in our study [27 (link)].