Alexa fluor plus 488 conjugated goat anti rabbit igg

Antibodies used were APC-conjugated CD133 antibody (catalog 130-113-184) from Miltenyi Biotec; PE-conjugated anti-CD24 (catalog 555428) from BD Biosciences; FITC-conjugated anti-EpCAM (catalog 60136FI) from StemCell Technologies; anti-SETD1A (catalog A300-289A) from Bethyl Laboratories; anti-PABPC1 (catalog A14872), anti-WDR5 (catalog A3259), anti-CXXC1 (catalog A13423), and anti-ASH2L (catalog A4892) from Abclonal; anti-H3K27ac (catalog ab4729) and anti-H3K27me3 (catalog ab6002) from Abcam; anti–β-actin (catalog AC004) from Abclonal Technology; anti–E-cadherin (catalog 3195S), anti-Vimentin (catalog 5741S), and anti-H3K4me3 (catalog C42D8) from Cell Signaling Technology; and Alexa Fluor Plus 488–conjugated goat anti-rabbit IgG (catalog A32731) and Alexa Fluor 594–conjugated donkey anti-rabbit IgG (catalog A-21207) secondary antibodies from Thermo Fisher Scientific. The detailed information for antibodies is shown in Supplemental Table 3. DAPI (catalog D9542) was from MilliporeSigma. B27 (catalog A3582801) and N2 supplements (catalog 17502001) were from Gibco. bFGF (catalog 157AA) was from Novoprotein, and EGF (catalog 236-EG) was from R&D Systems.

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.