Dab chromogenic kit

NOS2 antibody (Biogot, WB dilution 1:500, IHC-P dilution 1:100), ALOXE3 antibody (Biogot, WB dilution 1:1000), ALOXE3 (Bioss, IHC-P dilution 1:200), β-actin antibody (Biogot, dilution 1:5000), tissue protein extraction kit (Bestbio), animal tissue RNA stable preservation solution (Beyotime), BCA protein concentration assay kit (Beyotime), high-sensitivity ECL chemiluminescence kit, universal SP kit (ZSGB-BIO), DAB chromogenic kit (ZSGB-BIO).

Paraffin-embedded endometrial tissue was cut into 6 um thick slices, dewaxed twice in xylene for 20 min each time, and rehydrated. The sections were then treated with 3% H2O2 for 30 min to inactivate endogenous peroxidase and washed with PBS. The sections were placed in a pressure cooker containing Tris-EDTA (pH 9.0) and boiled for 2 min, after which the heat source was removed, and the sections were left at room temperature for 15 min. The lid of the pressure cooker was then opened and cooled to room temperature. The sections were blocked with a blocking buffer for 30 min at a temperature of 37 °C. Next, the sections were incubated with primary antibody at 4 °C overnight. The following day, the sections were incubated with goat anti-rabbit IgG H&L (HRP) (1:200, Abcam, Cambridge, UK) for 30 min at 37 °C. After washing thrice with PBS, the sections were stained using a DAB chromogenic kit (ZSGB-BIO, Beijing, China) for 5 min. The sections were then rinsed with running water, stained with hematoxylin (Solarbio, Beijing, China) for 6 min, fractionated with 1% hydrochloric acid for 30 s, dehydrated with gradient alcohol, made transparent with xylene, and sealed with a water-soluble sealer (ZSGB-BIO, Beijing, China). The positive area of the related proteins was measured by Image J software, and three images were used for statistical analysis.

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.