Hbred080cs01

IF and IHC assays were carried out as described previously [28 (link)]. The breast cancer tissue microarray (#HBreD080CS01) was purchased from Shanghai Outdo Biotech Company. The images of staining were captured using Nikon fluorescence microscope system. The scores of IHC staining (0 to 12) were conducted according to the percentage of positively stained tumor cells and staining intensity.

Tissue arrays containing multiple human breast cancer and pericarcinomatous tissues (HBreD055CD01and HBreD080CS01) were obtained from Shanghai Outdo Biotech Company (Shanghai, China). The clinical breast cancer tissue microarrays and mice tissue sections were analyzed by IHC staining, as previously described [52 (link)]. According to the protocols of the two-step detection kit (PV-9001, ZSGB-BIO, Beijing, China), the tissue microarray (TMA) slides or tissue sections were incubated in an appropriate endogenous peroxidase blocker for 10 min at room temperature, followed by incubation with anti-FRMD3 antibodies overnight at 4 °C and subsequent incubation with response enhancer and enhanced enzyme-labeled goat anti-rabbit IgG polymer for 20 min at room temperature. A DAB Chromogenic Kit (ZLI-9017, ZSGB-BIO, Beijing, China) was used to detect antibody binding, and the reaction was stopped by immersing the TMA slides or tissue sections in running water once a brown color appeared. Finally, the TMA slides or tissue sections were counterstained with hematoxylin (G1121, Solarbio, Beijing, China), dehydrated using a series of graded alcohol solutions, and mounted. Images were photographed with an Olympus BX50 microscope. Appropriate positive and negative controls were included for each run of the IHC assay.