Ab52587

Tissue slices were stained with hematoxilyn and eosin (HE) and tumor tissue vitality was confirmed. Slices with necrosis >50% were excluded from further immunohistochemical stainings. Slices were stained with antibodies against Ki67 (MIB-1, Dako, Glostrup, Denmark), PD-L1 (ab213524, Abcam, Cambridge, UK) or double stained using the Envision G/2 Doublestain System or Envision Flex Doublestain System (Dako). The antibody combinations were CD3 (IR503, Dako) + Ki67 (MIB-1, Dako), CD3 (IR503, Dako) + CK AE1/3 (IR053, Dako), CD8 (IR623, Dako) + Ki67 (MIB-1, Dako), PD1 (ab52587, Abcam) + Ki67 (MIB-1, Dako), and PD1 (ab52587, Abcam) + CK AE1/3 (IR053, Dako). All slides were stained with automatized immunostainers (Autostainer Plus, Dako).

IHC was carried out on formalin-fixed, paraffin-embedded tissue blocks according to the manufacturers’ instructions. IHC antibodies included those targeting p63 (clone 4A4, 1:200; Biocare), thyroid transcription factor-1 (TTF1) (clone 8G7G3/1, 1:50; Abcam), CK7 (EPR17078, 1:1000; Abcam), Napsin A (clone IP64, 1:200; Leica), PD-1 (NAT105; 1:100; ab52587; Abcam), PD-L1 (clone 28–8; 1:100; ab205921; Abcam) and PD-L1 (SP142; 1:100; ab228462; Abcam). IHC was performed as previously described [13 (link)]. The results were evaluated by two experienced pathologists who were blind to the patients’ clinical features. For p63, TTF1, CK7, and Napsin A, a positive score was based on moderate to strong staining observed by experienced pathologists.
For PD-1 and PD-L1, the expression was assessed in both tumor and stromal cells. Positive PD-1/PD-L1 expression was defined as staining of the cell membrane. PD-1/PD-L1 was validated quantitatively at specified levels of < 1%, 1–5%, 5–10% or ≥ 10% of tumor or stromal cells in a section that included at least 100 tumor or stromal cells. Since the expression of PD-1 was extremely low in tumor cells, the expression of PD-1 in tumors was grouped into two categories: < 1% and ≥ 1%.

Slides were deparaffinized and rehydrated in phosphate buffered saline solution (10 mM, pH 7.2). The tissue epitopes were demasked using the automated water bath heating process in Dako PT Link (Dako, Glostrup, Denmark); the slides were incubated in TRIS-EDTA retrieval solution (10mM TRIS, 1mM EDTA pH 9.0) at 98°C for 20 minutes. The slides were subsequently incubated for 1 hour at room temperature with the primary mouse monoclonal antibody against PD-1 (Abcam, [NAT105]: AB52587) and rabbit monoclonal antibody against PD-L1 (Abcam [EPR1161(2)]: AB174838) diluted 1:100 in Dako REAL antibody diluent (Dako, Glostrup, Denmark) and immunostained using anti-mouse/anti-rabbit immuno-peroxidase polymer (EnVision FLEX/HRP, Dako, Glostrup, Denmark) for 30 minutes at room temperature, according to the manufacturer's instructions. Color reaction was developed with diaminobenzidine substrate-chromogen solution (DAB, Dako, Glostrup, Denmark) for 5 minutes. Finally, the slides were counterstained with haematoxylin, mounted and reacted for 5 minutes with diaminobenzidine substrate-chromogen solution (DAB, Dako, Glostrup, Denmark) for visualization. PD-1 and PD-L1 positivity of lymphocytes in the tonsil was used as a positive control, same tissue with omitting of the primary antibody served as negative control.