Kit 9710

All the 271 tissues were embedded in paraffin, and the representative locations were marked by H&E staining. Tissue sample (1.5 mm) was extracted from each representative location and accurately transferred by the Organization Microarrayer (Pathology Devices, San Diego, CA, USA) to another paraffin block to establish the TMA. TMA sections of 4 µm thickness were adhered to poly-L-lysine-coated glass slides and prepared for IHC.
The microarray sections were deparaffinized with xylene for 30 minutes and hydrated in graded ethanol and distilled water. The microarray sections were placed in 0.01 M citrate buffer (pH 6.0), and antigen retrieval was performed for 8 minutes at a high pressure of 80 kPa. The sections were then treated with 3% H₂O₂ for 10 minutes at room temperature to remove endogenous peroxidase. After blocking the nonspecific protein-binding site with normal goat serum, rabbit antihuman ACTL8 polyclonal antibody (1:50, ab36030; AbSci, Vancouver, WA, USA) was added and incubated at 4°C overnight. On the next day, followed by incubation with biotinylated goat anti-rabbit IgG and streptavidin–horseradish peroxidase complex (KIT-9710; MXB biotechnologies, Fuzhou, Fujian, China) at 37°C for 30 minutes, the sections were stained with 3,3′-diaminobenzidine for 25 seconds. PBS replaced the anti-ACTL8 primary antibody as a negative control.

Paraffin-embedded tumor tissues were sliced into 5-μm thick sections and mounted on glass. Slides were deparaffinized and rehydrated in 10 minutes through a graded alcohol series to deionized water in 1% Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) and microwaved to enhance antigen retrieval. Tissue samples were sequentially incubated with anti-mouse immunoglobulin coupled to horseradish peroxidase. Slides were incubated with the specific primary mouse anti-human EGFR (MAB-0196; MXB, Fuzhou, China) and mouse anti-human HER-2 monoclonal antibodies with a dilution 1:200, then incubated with an horseradish peroxidase-conjugated secondary antibody (KIT-9710; MXB), and then stained with 3,3-diaminobenzidine and counterstained with hematoxylin. In addition, slides stained without primary antibody or without secondary antibody were used as the control. Two pathologists independently observed and interpreted the results of the immunohistochemical staining.

Deparaffinized 4-µm thick sections of the primary gastric cancers were rehydrated through a series of graded ethanol solutions and incubated for 10 min at RT with 3% H₂O₂. And washed in 0.01M PBS buffer (pH 7.4) at RT three minutes for three times, the sections were incubated with biotinylated NPL (Vector, Burlingame, CA, USA) at PBS buffer-diluted concentration of 1:200 at RT for 30 min. The sections were blocked with normal non-immune animal serum at RT for 15 min. After carefully washed in PBS buffer, the sections were incubated with a streptavidin-peroxidase complex (KIT-9710, MXB Biotechnologies, Fujian, China) at RT for 10 min. Finally, all the sections were visualized with DAB kit (DAB-0031, MXB Biotechnologies, Fujian, China), and counterstained with hematoxylin.