Ab228462

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%.

Tissue samples were collected and stained followed the manufacturer’s protocol. In brief, deparaffinized and rehydrated sections were treated for antigen retrieval using sodium citrate buffer, blocked with normal goat serum for 30 min at room temperature, and then incubated with primary antibody against CD3 (ab16669, Abcam, Cambridge, UK), PD-L1(ab228462, Abcam), Ki67(D2H10, Cell Signaling Technology, Danvers, MA) at 4 °C overnight. Slides were incubated with secondary antibodies, counterstained with hematoxylin, and visualized by the Ultra Vision Detection System (Thermo Fisher Scientific). Signal intensity was scored by two independent observers who were blind to the experimental groups.

The DGs tissues were fixed, dehydrated, and paraffin-embedded. The 4 μm sections were deparaffinized and rehydrated in 100, 95, and 75% ethanol. The antigen retrieval solution with EDTA (pH 9.0) was used for PD-L1 staining, while the solution with sodium citrate (pH 6.0) for B7-H3 and CD47 staining. After endogenous peroxidase activity blocking, the sections were rinsed and incubated with the primary antibodies including anti-human PD-L1 (1:1,000, Abcam, ab228462, Cambridge, MA, USA), CD47 (1:2000, Abcam, ab218810) and B7-H3 (1:2,000, Abcam, ab219648) overnight at 4°C. TILs were stained by the CD45 (1:1,000, Abcam, ab40763) antibody, and TAMs were stained by the CD68 antibody (1:1,000, Abcam, ab213363). Images were acquired after the incubation with secondary antibodies and DAB, and counterstained with Hematoxylin. The expression level of three immune checkpoint molecules was determined by the percentage of positive cells and the staining intensity: low (negative intensity and intensity 1, and intensity 2 with positive cells < 10%) and high (intensity 2 with positive cells ≥ 10% and intensity 3) expression (19 (link)). The expression of TILs/TAMs was similarly evaluated as previously described (20 (link), 21 (link)).