Anti pd l1 clone 28 8

Well-characterized anti-PD-L1 (clone 28–8; Abcam, Cambridge, MA, USA) and anti-cluster of differentiation 68 (CD68) (clone KP1; DAKO, Glostrup, Denmark) antibodies were selected. IHC was performed using an automated staining system (Leica Bond III; Leica Microsystems). The antibody dilutions were optimized to 1:100 for anti-PD-L1 and 1:400 for anti-CD68. The slides were dewaxed and rehydrated using distilled water, and were subsequently processed for PD-L1 (heat-induced antigen retrieval at pH 9.0) or CD68 (proteolytic treatment). After incubation with the primary antibodies (anti-PD-L1, 30 minutes; anti-CD68, 15 minutes), the tissue sections were rinsed, and the sections for PD-L1 staining were further incubated with EnVision FLEX+ Rabbit LINKER (DAKO) and EnVision+ HRP Labelled Polymer (DAKO). The sections for CD68 staining were incubated with the Bond Polymer Refine Detection Kit (Leica Microsystems). Staining was visualized using diaminobenzidine, and counterstaining was performed using hematoxylin. Formalin-fixed, paraffin-embedded tissue blocks of human placenta and tonsil were prepared as positive controls. The stained slides were scanned as whole-slide images using a ScanScope^® Aperio CS2 slide scanner (Leica Microsystems).

IHC staining was performed on 4 μm formalin-fixed paraffin-embedded tissue sections, which were incubated at 60 °C for 1 h and deparaffinized and rehydrated with xylene and graded alcohol. For antigen retrieval, the sections were performed with EDTA (pH 8.0) or citrate buffer (pH 6.0) at high temperature and pressure for 3 min and cooled down to room temperature. Tissue sections were blocked with 3% hydrogen peroxide methanol solution for 30 min at 37 °C to inactivate the endogenous peroxidase and then added with animal nonimmune serum for 30 min in a humid chamber at 37 °C, followed by incubation with the primary antibodies overnight at 4 °C, including anti-ATAD3A (NBP2-14881, Novus Biologicals), anti-PD-L1 (clone 28-8, Abcam; D5V3B, Cell Signaling Technology), anti-CD8a (D4W2Z, Cell Signaling Technology), anti-CD8 (clone C8/144B, MXB Biotechnologies), anti-granzyme B (D6E9W, Cell Signaling Technology), anti-Ki67 (clone SP6, Abcam). After washing with PBS three times, the sections were incubated with secondary antibody (Dako) for 30 min in a humid chamber at 37 °C. The sections were washed with PBS for three times and performed with Dako Real™ Envison kit for staining. IHC score was quantified using Image-Pro Plus software 6.0, determined by integrated optical density (IOD) value/area, three to five fields per section.