Kit 9707

Xenograft tumor tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned with a semiautomated rotary microtome (Leica, RM2235). Morphology was evaluated by hematoxylin and eosin (H&E) staining. The protein levels of H3K9Ac and Ki67 were analyzed by immunohistochemistry according to standard protocols with specific primary antibodies (anti-H3K9Ac, 1:100 dilution, ab32129, Abcam; anti-Ki67, 1:200 dilution, ab15580, Abcam). All assays were followed by staining with HRP-conjugated rabbit secondary antibody (KIT-9707, Maixin Biotechnology), and samples were then counterstained with hematoxylin and mounted. Images were captured with a Nikon Ti-S microscope.

The protein levels of Cbx4 and VEGF in cells of tumor tissues were analyzed by IHC, respectively with anti-Cbx4 antibody (1 : 25 dilution, SC-19299, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-VEGF polyclonal antibody (1 : 500 dilution, SC-152, Santa Cruz Biotechnology). Angiogenesis was assessed using IHC with anti-CD31 antibody (1 : 50 dilution, Gene Tech, Shanghai, China). All primary antibody stainings were followed by staining with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (KIT-9719 and KIT-9707, Maixin Biotechnology, Fuzhou, China). IHC scores of Cbx4 and VEGF were divided into two classifications: low (immunoreactive scores ≤4) and high (immunoreactive scores >4), according to the value of IRS systems as described in our previous work.^{34 (link), 42 (link)} At × 200 magnification, vessel count was made of all distinct brown-staining endothelial cells in the cancerous regions over five fields in each slide. MVD was defined as the average value of three readings. Angiogenesis status was assessed by MVD classification: low (≤50/ × 200 magnification) and high (>50/ × 200 magnification), according to the mean MVD of cancerous-tissue vessels. Images were captured with Nikon Ti-S microscope equipped with a digital camera system (Nikon, Tokyo, Japan).