Pv 6000

For GC tissue, paracancer tissue, and greater omentum metastasis tissue, paraffin sections were firstly dewaxed, antigen repair, peroxidase block, and sheep serum block. Then choose the primary antibody, ADORA2B (1:100, ER1903-44, HUABIO, China), β-catenin (1:200, ab32572, Abcam, USA), N-cadherin (1:200, ab18203, Abcam, USA), vimentin (1:100, 10366-1-AP, Proteintech, China) and E-cadherin (1:200, ab40772, Abcam, USA) incubated overnight in a 4°C humidor. For immunohistochemical detection, secondary antibody (PV6000, ZSGB-BIO, China) was added and incubated at 37°C for 20min. Then DAB was performed. For immunofluorescence, sections were cleaned and goat antirabbit antibody combined with secondary antibody Alexa 488 was incubated for 1h. The nuclei were restained with DAPI.

The expression of KIF23 was detected in paraffin-embedded tumor tissue by a rabbit-anti-KIF23 antibody (ab235955, 1:200 dilution; Abcam, USA), following the procedure specified in the immunohistochemistry kit (ZSGB-BIO, pv6000, China). Briefly, we fixed tumor tissues and matched normal adjacent tissues in 10% neutral-buffered formalin and embedded them in paraffin blocks by the pathology department of our hospital. Then, the paraffin sections (4 µm) were placed into a microwave oven for 15 minutes to repair the antigens and were then cooled at room temperature. Endogenous peroxidase was blocked, and sections were incubated with an anti-KIF23 antibody overnight at 4 °C, followed by a goat anti-rabbit secondary antibody at 37 °C for 1 hour. Sections were stained with 3,3-diaminobenzidin (DAB) at room temperature for 10 minutes and images were then recorded by microscopy (Olympus BX43). All samples were evaluated by two independent pathologists.
To analyze the results, KIF23 nuclear staining was scored using the 4-point scale (0, no staining; 1+, light staining at high magnification; 2+, intermediate staining; and 3+, dark staining of the linear membrane at low magnification). According to the distribution of scores of KIF23 staining intensity, samples were divided into high expression (2+ to 3+) and low expression (0 to 1+) groups.

The left lung tissue was fixed in 10% buffered formalin for 48 h. Tissue samples, from paraffin-wax-embedded blocks, were sectioned to be 4–5 µm thick. Following the process of dewaxing, hydration, closed endogenous peroxidase and antigen repair, polyclonal IL-21 antibody (rabbit anti-mouse, 1:300) and universal secondary antibody (PV6000, ZSGB-BIO, Beijing, China) were applied (1–2 h at room temperature). Slides were observed using a light microscope at a magnification of ×200 and ×400. Sections were considered positive according to the color observation, which is an indication of the antibody-antigen reaction, and manifested as an intracytoplasm brown coloration in different areas of the stained tissue section.