Ab60076

Intratumoral T-cell subpopulations were detected by a combination of primary antibodies: anti-CD3 (Dako, A0452, Host-rabbit), anti-CD8 (Clone YTC182.20, Abcam, Ab60076, Host-rat), and anti-FOXP3 (Clone 236 A/E7, Abcam, Ab20034, Host-mouse) on acetone-fixed breast tumor cryosections as described earlier [27 (link)]. Primary specific secondary antibodies (anti-rabbit Alexafluor 647, A21245; anti-rat Alexa Fluor 488, A11006; anti-mouse Alexa Fluor 555, A31570) were purchased from Life Technologies. DAPI (Invitrogen, D1306) was utilized to stain cell nuclei.
Total tissue slides were scanned on Olympus IX51 microscope equipped with a F-View II camera (both Olympus) and analyzed by the TissueQuest Cell Analysis Software package (version 4.0.1.0137, TissueGnostics GmbH). For automated analysis with TissueQuest, DAPI staining was used as a master marker for cell identification on the basis of nuclei detection. Based on H&E stained reference slides, regions of interest (ROI) were defined to distinguish between tumor and surrounding non-tumor area. All tissues were analyzed with identical parameters for detection of T cells based on nuclear size, mean staining intensity, and background threshold. Cells were visualized in scattergrams, while the cutoff between positive and negative gated cells was validated manually by backward gating on the original image.

For immunofluorescence analysis, tissues were stained with CD8 antibody (ab60076, abcam, Cambridge, USA), PD1 antibody (ab137132, abcam), SALL4 antibody (ab29112, abcam), hepatocyte specific antigen antibody (GTX73779, Genetex), CD45 antibody (MA5-17687, Thermo Scientific, Waltham,USA) and PD-L1 (ab205921, abcam) followed by Alexa Fluor 647 conjugated goat anti-rat IgG (A-21247), Alexa Fluor Plus 488 conjugated goat anti-rabbit IgG (A-32731) and Alexa Fluor Plus 555 conjugated donkey anti-mouse IgG (A-32727) (Thermo Scientific). Images were acquired on a Zeiss 710 Meta multi-photon confocal microscope (Zeiss, Oberkochen, Germany). To assess the immunostaining quantification, we analyzed the slides via an image analysis workstation (Image Pro Plus 6.0, Media Cybernetics). Mouse liver tissues were collected and embedded in OCT. Intrahepatic HBsAg, Pd-l1, or Sall4 expression was visualized by immunohistochemical staining with rabbit anti-mouse HBs Ab (Genetech, Shanghai, China) or rabbit anti-mouse Pd-l1 Ab (eBioscience, San Diego, CA, USA), or anti-Sall4 Ab (abcam, cat#57577) followed by Envision System HRP detection staining (Genetech, Shanghai, China) performed according to the manufacturer’s protocol. Liver sections were stained with hematoxylin. Images were taken with an OLYMPUS microscope.