Diaminobenzidine zli 9018

Tumor tissues were fixed in 4% paraformaldehyde, processed through a series of dehydration steps, and embedded into paraffin blocks. Paraffin-embedded tissues were cut into 4 μm slices and adhered on adhesive slides. Tissue sections were stained with hematoxylin–eosin. For immunohistochemistry, sections were de-waxed, antigen-repaired, and blocked for nonspecific reactions. Then, the sections were incubated with anti-MARCH1 (1:200, #YT2642, Immunoway, Newark, United States) or anti–Ki-67 (1:200) antibody for overnight at 4 °C and then incubated with secondary antibody (#PV-6000, ZSGB-BIO, Beijing, China) for 30 min at 37°C. Diaminobenzidine (#ZLI-9018, ZSGB-BIO, Beijing, China) was used for color development. The sections were imaged with a light microscope (Leica Microsystems, Wetzlar, Germany). Quantification of section staining was performed by Image-Pro Plus 6.0.

CIPp xenograft tumors were dissected and fixed with10% (v/v) neutral-buffered formalin (G2161, Solarbio, China), embedded in paraffin wax and sectioned serially at 3 μm. After deparaffination and antigen retrieval with EDTA Antigen Retrieval Solution (G203, Epsilon, China), the tumor sections were incubated overnight at 4 °C with primary antibody for the proliferation marker protein antigen identified by monoclonal antibody Ki-67 (Ki67) (27309–1-AP; Proteintech, China, 1:1000). The biotinylated secondary antibody, anti-rabbit antibody IgG (ZB-2010, ZSGB-BIO, China), was incubated for 1 h at room temperature. Then the sections were stained with diaminobenzidine (ZLI-9018, ZSGB-BIO, China) and counterstained with hematoxylin (C0107, Beyotime, China). Images were captured with a digital microscope, and the amounts of Ki67 positive cells and total cells per image were automatically calculated by color using Image-pro-plus (IPP) software (Media Cybernetics, Washington, USA). The ratio between Ki67 positive cells and total cells was defined as the percentage Ki67 positive cells.