Two step detection kit

IHC staining of Ihh was performed using a Two-step Detection Kit from ZSGB-BIO (#PV-9001, Beijing, China). Briefly, cryosections of lower lumbar IVDs from 2-week-old mice were air-dried at RT, and incubated with 10 mM citric acid solution (pH 6.0) for 4 h at 60 °C for antigen retrieval. Subsequently, sections were blocked with endogenous peroxidase blocker (ZSGB-BIO), followed by incubation with Ihh primary antibody (1:100; Abcam; ab52919) for 60 min at 37 °C. Sections were incubated with reaction enhancement solution (ZSGB-BIO) for 60 min at RT and then incubated with enhanced enzyme-conjugated goat anti-rabbit IgG for 15 min at RT. After three washes with PBS, sections were stained with DAB reagent (ZLI-9019, ZSGB-BIO) and then counterstained with hematoxylin. Afterwards, sections were dehydrated with gradient alcohol, cleared with xylene, and finally sealed with neutral resin.

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.