Arg52748

Immunohistochemistry was undertaken with antibodies against CNBP (sc‐515387, dilution: 1:200), Ki‐67 (sc‐23900, Santa Cruz Biotechnology, Inc., dilution: 1:200), CD31 (Arigo, ARG52748, Arigo, Hsinchu City, Taiwan, 1:100 dilution), CD3 (ab135372, dilution: 1:200) or F4/80 (ab16911, Abcam Inc., dilution: 1:200). Using an Olympus BX43 microscope (Tokyo, Japan), 10 distinct fields of high power (400×) were viewed for each specimen, while positive cell percentage was evaluated via Image‐Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA).⁴⁵, ⁴⁷, ⁴⁹

The experimental detection steps were conducted according to the instructions described in Immunohistochemistry (Boster Bio, China). After dewaxing, the paraffin section was incubated with 3% H₂O₂ at room temperature for 10 min. The samples were washed five times with PBS and the slices were subjected to antigen thermal repair in an antigen repair solution. The paraffin section was incubated with 5% BSA solution for 30 min at 37°C. After incubation of the anti-CD31 (dilution, 1:50; ARG52748, Arigo Biolaboratories, Shanghai, China), HK2 (dilution, 1:500; ab209847, Abcam, Cambridge, UK), c-Myc (dilution, 1:200; ab32072, Abcam, Cambridge, UK), Ki67 (dilution, 1:200; 9449, Cell Signaling Technology, Boston, USA), E-cadherin (dilution, 1:400; 3195, Cell Signaling Technology, Boston, USA), and Vimentin (dilution, 1:200; 5741, Cell Signaling Technology, Boston, USA) antibodies overnight at 4°C, the paraffin section was incubated with HRP-labeled anti-rabbit/mouse IgG for 30 min at 37°C. DAB was used for color rendering under a microscope, while hematoxylin was employed for staining. The images were collected using a microscope (Ti-S; Nikon, Tokyo, Japan).

Immunohistochemical staining and quantitative evaluation were performed as previously described (Zhang et al, 2012; Zhao et al, 2016; Li et al, 2018a,b), with antibodies specific for Ki‐67 (1:500, sc‐23900, Santa Cruz Biotechnology) or CD31 (1:500, ARG52748, Arigo, Hsinchu City, Taiwan). The degree of positivity was blindly assessed by at least two pathologists.