Dako automation hematoxylin

The paraffin tissue sections were incubated at 60 °C for 1 h, then loaded onto the Dako autostainer and treated with Proteinase K for 5 min. Subsequently, the Dako autostainer applied the antibodies (PD-L1, 22C3, Dako, Santa Clara, USA), and slides were incubated at room temperature for 30 min. Following a buffer wash, slides underwent a 30-min incubation with the labeled polymer, HRP, at room temperature. The DAB⁺ substrate-chromagen solution was then applied for 10 min. Counterstaining was carried out with Dako automation hematoxylin for 5 min, followed by post-counterstaining with a bluing agent for 1 min. After washing, slides were dehydrated in three sequential washes of 70%, 95%, and 100% reagent alcohol, and three xylenes baths before coverslips were applied. Binary scoring, utilizing the FDA-approved companion assay cut-point for PD-L1–stained tumor-infiltrating ICs (of any staining intensity, covering ≥ 1% of the tumor area), was conducted by two board-certified. Anatomic pathologists specialized in PD-L1 interpretation. In cases of discordance, a consensus-based final PD-L1 score was assigned after re-review.