D5v3b

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.

After fixation for at least 24 h in 4% PBS-buffered PFA the left lung lobe was serially cut into 4 µm sections and stained with hematoxylin and eosin to assess metastasis. The metastatic area was evaluated with QuPath 0.4.3. Tumors were fixed similarly and immunohistochemistry was subsequently performed for CD8α (1:100, 4SM15-Biotin, eBioscience, San Diego, CA, USA) with secondary detection by Streptavidin-AP (1:100, Invitrogen, Waltham, MA, USA), anti-BrdU (1:50, BU20a, Dako, Hamburg, Germany) with secondary detection by a HRP-conjugated antibody (polyclonal goat anti-mouse, 1:100, Dako, Hamburg, Germany) or anti-PD-L1 (1:200, D5V3B, Cell Signalling Technology, Danvers, MA, USA) with secondary detection by an AP-conjugated antibody (goat anti-rabbit, 1:200, 97048, Abcam, Waltham, MA, USA). Detection of BrdU-positive (BrdU⁺) and CD8-positive (CD8⁺) cells as well as PD-L1-positive tissue was performed with QuPath 0.4.3.

Immunohistochemistry was performed on 5-µm thick sections of formalin-fixed paraffin-embedded (FFPE) NA13 tumors blocks using a Rabbit anti-mouse antibody (D5V3B, Cell Signaling Technology, USA) at a dilution of 1:100. Antigen retrieval was performed according to standardized protocols by heating with 10 mM citrate buffer. The activity of endogenous peroxidase (peroxidase blocking reagent, Dako) were neutralized and non-specific binding were then blocked with goat serum 1:100 in PBS. Tissue sections were incubated with primary antibodies. The slides were then incubated with anti-rabbit HQ secondary antibody followed by anti-HQ HRP linking antibody (Ventana Medical System, USA). Finally, slides were incubated with Discovery ChromoMap DAB reagent. Slides were counterstained with hematoxylin, dehydrated by ethanol and mounted. Slides were imaged on an Olympus BX43 light microscope with ×20 magnification using an Olympus DP26 digital camera.