Alexa fluor 647 conjugated goat anti rat igg

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.

Immunofluorescence was performed according to our previously described method [16 (link)]. Brain tissues were resected from the mice, fixed in 4% paraformaldehyde for 24 h, dehydrated with 30% sucrose solutions, and frozen in compound. The sections (40-μm thick) were blocked with 0.1% Triton X-100 and 5% normal donkey serum in PBS for 20 min, and then incubated with the following primary antibodies overnight at 4°C: anti-F4/80 (1:200; Abcam, Cambridge, UK) and anti-CD3 (1:200, Abcam). The sections were then incubated with a mixture of Alexa Fluor 647-conjugated goat anti-rat IgG (1:1000; Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000; Thermo Fisher Scientific) antibodies for 30 min at 37°C. Next, 4′,6-diamidino-2-phenylindole (DAPI, 1:3000; Sigma-Aldrich) was applied for 5 min, after which the samples were washed three times in PBS for 15 min. The sections were then mounted on SuperFrost slides, and all images were captured using a confocal fluorescence microscope (TCS-TIV; Leica, Nussloch, Germany).

Immunostaining and alkaline phosphatase (AP) staining were performed as described previously [15 (link)]. Primary antibodies were anti-stage-specific embryonic antigen 1 (SSEA1, MC-480; Developmental Studies Hybridoma Bank, DSHB, Iowa City, IA), anti-Oct3/4 (MBL, Nagoya, Japan), anti-nestin (Rat-401; DSHB), anti-α-smooth muscle actin (Progen, Heidelberg, Germany), anti-neuronal class III β-tubulin (Tuj1; Covance, Richmond, CA), anti-Sox2 (Millipore, Bedford, MA), anti-glial fibrillary acidic protein (GFAP; Dako, Carpenteria, CA), anti-α-actinin (Sigma-Aldrich, Tokyo, Japan), anti-α-fetoprotein (R&D Systems, Minneapolis, MN), anti-Sox1 (Cell Signaling Tech, Beverly, MA), anti-Sox10 (Millipore), anti-PGP9.5 (Ultra Clone, Wellow, Isle of Wight, UK), and anti-GFP (Nakarai Tesque, Kyoto, Japan). Secondary antibodies were Alexa Fluor 568-conjugated goat anti-mouse IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG1, Alexa Fluor 568-conjugated goat anti-mouse IgM, Alexa Fluor 647-conjugated goat anti-mouse IgG, Alexa Fluor 647-conjugated goat anti-rat IgG, and Alexa Fluor 488-conjugated goat anti-rabbit IgG (all purchased from Molecular Probes, Life Technologies, Eugene, OR).