Anti pd l1 d5v3b

Tumors grown in C57BL/6 mice were harvested 7 days following treatment, zinc-fixed, sectioned and prepared as previously described [43 (link)]. Primary antibodies were anti-CD3 (SP7, Abcam, Cambridge, UK), anti-CD8α (4SM15, eBioscience), anti-FoxP3 (FJK-16s, Invitrogen, Carlsbad, CA, USA), anti-PD-L1 (D5V3B, Cell-Signaling Technology, Danvers, MA, USA), and DAPI (Perkin Elmer). Anti-Rat HRP (mouse adsorbed) or Anti-Rabbit HRP secondary antibodies (Vector Laboratories) were used prior to tyramide staining with Opal 7-Color Automation IHC Kit (690 for CD3, 570 for PD-L1, 620 for CD8α, and 520 for FoxP3). Slides were scanned with Vectra Polaris (Perkin Elmer) and analyzed using QuPath 0.2.0-m4 [44 (link)].

Tumors grown in C57BL/6 and C3H mice were harvested upon reaching 12 mm in diameter and were fixed in zinc-based fixative for 24 hours at room temperature as previously described (16 (link)). Tissue was then processed by preparing 4 µm thick tissue sections, incubation of slides at 37°C and deparaffinization. The protocol included blocking with goat serum (Vector) prior to staining and dilution of primary antibodies in Renaissance Background reducing Diluent (Biocare medical). Tissue sections were boiled in Rodent Decloaker (Biocare Medical) for antigen retrieval, except prior to CD8 staining where pH=9 buffer (Perkin Elmer) was used. Primary antibodies were anti-CD3 (SP7, Abcam, Cambridge, UK), anti-CD8α (4SM15, eBioscience), anti-FoxP3 (FJK-16s, Invitrogen, Carlsbad, CA), anti-PD-L1 (D5V3B, Cell Signaling Technology, Danvers, MA), and DAPI (Perkin Elmer). Opal 7-Color Automation IHC Kit was used (690 for CD3, 570 for PD-L1, 620 for CD8α, and 520 for FoxP3). Slides were scanned with Vectra Polaris (Perkin Elmer, Waltham, MA) and analyzed using QuPath 0.2.0-m4 (17 (link)).

Anti-phospho-Stat1 (Tyr701) (58D6), anti-phospho-Stat3 (Thyr705) (D3A7), anti-phospho-iκB (14D4), anti-iNOS (D6B6S), and anti-PD-L1 (D5V3B) were purchased from Cell Signaling. Anti-CD3, anti-CD28, anti-CD25, anti-CD44, anti-CD62L, anti-IFN-γ, IL-6, and anti-TNF-α were purchased from BD Biosciences. Anti-Foxp3 antibody was purchased from eBioscience.