Anti cd3 sp7

The mouse livers were fixed with 10% formalin, sectioned, and stained with hematoxylin and eosin (H-E). Ki67, CD3, F4/80, and Gr-1 immunostaining was performed using anti-Ki67 (SP6, NeoMarkers, Fremont, CA, USA), anti-CD3 (SP7, Abcam, Cambridge UK), anti-F4/80 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or Gr-1 (eBioscience, San Diego, CA, USA) Abs, respectively, and a VECTASTAIN Elite ABC Kit. Diaminobenzidine tetrahydrochloride (DAB) was used as the peroxidase substrate and sections were counterstained with hematoxylin. The positive immunostained area was analyzed using ImageJ software (National Institutes of Health [NIH], Bethesda, MD, USA; http://rsb.info.nih.gov/ij/) and the percentage of positive-stained tissue relative to the total area of the tissue section was determined.

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)).

TILs were assessed using H and E slides in FFPE sections obtained through core needle biopsies prior to therapy and through surgical specimens after treatment at surgery. Separate individual slides were stained with anti-CD56 (123C3.D5 + 123A8, Abcam plc, Cambridge, MA), anti-CD3 (SP7, Abcam plc), anti-CD8 (SP16, Abcam plc), anti-FoxP3 (1054C, R and D systems), and anti-CD68 (KP1, Abcam plc) antibodies. All slides were prepared and stained by the UAMS pathology core. The percentage of TILs was determined according to the method proposed by the International Immuno-Oncology Working Group [31 (link), 32 (link)]. The same strategy was used to score various lymphocytes including CD56- and CD3-positive TILs. Slides were independently evaluated by two pathologists (SPO and GRP). Images were analyzed using a Nikon Eclipse Ni microscope at 20X magnification. Photomicrographs were taken with a Nikon DS-Fi3 microscope camera using the NIS-Elements D software from Nikon.