Immunohistochemical Detection of Snail

Paraffin sections were deparaffinized, hydrated in a graded ethanol series, and then boiled for 15 min in citrate antigen retrieval solution (Solarbio). The sections were then stained using a universal streptavidin-perosidase staining kit (Solarbio) as previously described [22 (link)]. Briefly, the sections were treated with 0.3% H₂O₂ for 30 min at 37 °C to quench endogenous peroxidase activity, washed with PBS, and blocked with pre-immune serum for 30 min at 37 °C. Following the manufacturer’s instructions, the sections were incubated with an anti-Snail (1:100, diluted in PBS, Santa Cruz Biotechnology, Inc., Dallas, Texas, USA, sc-393172) specific primary antibody at 4 °C. The secondary antibody was incubated at 37 °C for 1 h and then incubated with streptavidin–biotin peroxidase for 30 min at 25 °C. Finally, the sections were visualized with diaminobenzidine lightly counterstained with hematoxylin, dehydrated, and mounted with coverslips. Sections were viewed under a microscope (Nikon, Co., Tokyo, Japan) after drying at 25 °C.