Zm 0166

Sections 4 μm in thickness were de-paraffinized and rehydrated by standard protocols. Antigen retrieval was performed by heating slides in retrieval buffers pH6 in a microwave. Sections were incubated in blocking buffer (goat serum) for 1 h at room temperature and followed by incubation overnight at 4°C with primary antibodies against Ki-67 (ZSGB-Bio, ZM-0166), GFP (Abcam, ab6556, dilution 1:500), TFPI2 (Abcam, ab186747, dilution 1:100), and endomucin (Ebioscience, 14-5851, dilution 1:1,000). Sections were incubated with the secondary antibodies (rabbit (PV-6001), mouse (PV-6002), and rat (PV-6004) polymer detection system, ZSGB-Bio) for 1 h at room temperature and then stained with DAB and counterstained with hematoxylin. Staining intensity scoring was performed as previously described (37 (link)). endomucin-PAS staining was performed after staining of endomucin with DAB. Sections were incubated with 0.5% periodic acid for 15 min, rinsed in distilled water for 10 min, Schiff’s reagent for 30 min, and counterstained with hematoxylin (38 (link)).

The pretreatment of samples for immunofluorescence was the same as for immunohistochemistry, antigen was retrieved by microwave heat treatment in citrate buffer (pH 6.0) for 25 min. and then penetrated with 0.3% Triton X-100 in PBS for 1 h and further placed in 5% blocking buffer (normal donkey serum, 017-000-021; Jackson) for 1 h at RT. Sections were then incubated with the appropriate primary antibodies in 5% donkey serum overnight at 4°C. After additional several washes in PBS, slides were incubated with secondary antibodies in the dark for 1 h at RT, and the nuclei were labeled with Hoechst 33342 (Thermo Fisher Scientific, H3570). Finally, Images were captured using ZEISS confocal laser scanning microscope LSM900 system. The following antibodies were used for immunofluorescence: anti-CD163 (Abcam, ab182422), anti-CD45 (Abcam, ab10558), anti-Ki67 (ZSGB-Bio, ZM-0166), anti-H3K9me3 (Abcam, ab8898). anti-OCT3/4 (Santa Cruz, sc-5279), anti-SOX2 (Santa Cruz, sc-17320), and anti-NANOG (Abcam, ab21624).