Zli 9067

Formalin-fixed, paraffin-embedded (FFPE) blocks of tumor tissue were sliced into 5 µm sections, deparaffinized in xylene (3×5 minutes) and then rehydrated through graded concentrations of ethanol (2×2 minutes in 100% ethanol, 1×2 minute in 95% ethanol, 1×2 minute in 75% ethanol). Tissue sections were then heated in EDTA solution (ZLI-9067; ZSGB-BIO, Beijing, China), for 150 seconds. After cooling, the sections were washed with PBS and then incubated with primary antibodies at 4°C overnight. The primary antibodies used were rabbit antibodies against CD34 (ZA-0550; ZSGB-BIO), CD99 (ZA-0577; ZSGB-BIO), Bcl-2 (ZA-0536; ZSGB-BIO), Ki67 (ZA-0502; ZSGB-BIO), and S100 (ZA-0225; ZSGB-BIO), as well as mouse antibodies against CK5/6 (ZM-0313; ZSGB-BIO) and CK-pan (ZM-0069; ZSGB-BIO). Slides were washed again and incubated with secondary antibodies using a PV-9000 kit (ZSGB-BIO) according to the manufacturer’s manual. The slides were then stained with hematoxylin (Absin, Fuzhou, China).

Skin tissues were fixed in 4% paraformaldehyde solution for 24 h. Sections were prepared after being embedded in paraffin. The slides were baked at 65°C for 2 h before deparaffinization, and then rehydrated in distilled water for 10 min. For antigen retrieval, slides were submerged in EDTA (pH 8.0; ZSGB-BIO, ZLI-9067) at 95°C for 20 min. After being blocked with immunol staining blocking buffer (Beyotime Biotechnology, P0260) at 25°C for 30 min, the slides were incubated with primary antibodies at 4°C overnight. Then, the slides were incubated with secondary antibody at 37°C for 1 h. Images were captured by a Nikon Ni-U camera (Nikon). Five fields were observed for each section. Scoring was conducted according to the immunoreactive score (IRS) standard as previously reported [91 (link)]. Primary antibodies used in the experiment are listed in Table 2.