Sp 9000

The expression of S100A9 in tissues was examined by IHC. The sections from the formalin fixed, paraffin-embedded tissues were deparaffinized and dehydrated. Then the sections were boiled for 10 min in 0.01 M citrate buffer and incubated with 0.3% hydrogen peroxide (H₂O₂) in methanol for 15 min to block endogenous peroxidase. The sections were then incubated with the anti-S100A9 polyclonal antibody (1:300 dilution; ab63818, abcam, UK) overnight at 4 °C, following incubation with secondary antibody tagged with the peroxidase enzyme (SP-9000, Zhongshan Golden Bridge, China) for 30 min at room temperature and were visualized with 0.05% 3,3-diamino-benzidine tetrachloride (DAB) till the desired brown reaction product was obtained. The sections were finally counter-stained with hematoxylin. Control sections were performed using phosphate buffer solution (PBS) without a primary antibody. All slides were observed under a Nikon E400 Light microscope and representative images were taken.

LFs were fixed in paraformaldehyde. The proteins, p-ERK1/2 and α-smooth muscle actin (α-SMA), were detected using standard streptavidin–peroxidase immunocytochemical staining procedures. Cells were incubated with primary antibody (#4370 for p-ERK1/2 and #56856 for α-SMA, both 1 : 100; Cell Signaling Technology, Boston, MA, USA) at 4°C overnight. The same procedure without the primary antibody was used as an isotype control. Cells were incubated with secondary antibody (SP-9000; Beijing Zhongshan Goldenbridge Biotechnology, Beijing, China) for 20 min at 37°C, followed by horseradish peroxidase (HRP-) labeled streptavidin at 37°C for 20 min. Cells were washed with PBS three times between steps. After diaminobenzidine (DAB) staining, LFs were observed using a MetaMorph Imaging System (Universal Imaging Corporation, West Chester, PA, USA). The optical density value was semiquantitatively measured using the MetaMorph software. Five randomly selected high-power fields (400x magnification) were photographed for each slide.

Immunohistochemical specimens were fixed in 10% formaldehyde, which were then embedded in wax blocks. This study obtained anti-DDX56 antibody in Santa Cruz Biotechnology (5A7: sc-101,018, Santa Cruz, CA) and Ki-67 polyclonal antibody in Proteintech Group (No. 26,593-1-AP, ProteinTech, Wuhan, China). Following manufacturer’s instruction, the endogenous peroxidase blocking reagent (SP-9000, Zhongshan Golden Bridge, Beijing, China), goat serum (SP-9000, Zhongshan Golden Bridge), anti‐DDX56 antibody, Ki-67 polyclonal antibody, goat anti-mouse IgG (SP-9000, Zhongshan Golden Bridge), goat anti-rabbit IgG (SP-9000, Zhongshan Golden Bridge), HRP-labeled avidin working fluid (SP-9000, Zhongshan Golden Bridge) and Diaminobenzidine (DAB; ZLI-9018, Zhongshan Golden Bridge) were added in a regular sequence. Finally, the tissue sections were stained, and then a light microscope (Olympus Corporation, Tokyo, Japan) was employed for observation. Tumor histology was independently reviewed by an experienced pathologist. The DDX56 levels were rated as 0–3, indicating negative, low, moderate, and high intensities, respectively. The scores regarding the extent of staining were 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). In addition, a score of >4 was suggested as high.