Dab color development kit

Four-μm-thick formalin-fixed, paraffin-embedded serial sections of lung tissues were used for immunohistochemistry analysis of E-cadherin and vimentin expression. The sections were immersed in xylene for 15 minutes before being rehydrated in water by using an ethanol gradient. Then the sections were immersed in citric acid (pH 6.0; DAKO) for 10 minutes, after the samples were cooled to room temperature, the sections were washed with water and PBS buffer for 15 minutes before incubated with 3% H₂O₂ for 10 minutes. Then the sections were blocked with 5% BSA for 30 minutes before incubated with the primary antibody overnight at 4 °C. Color development was performed by using a DAB color development kit (DAKO). Sections were scanned by Pannoramic Scanning Electron Microscope to view the images.

Clinical tumor samples were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm sections. Sections were deparaffinized, rehydrated, and the antigens were retrieved with EDTA epitope retrieval buffer (pH 8.0). Horseradish peroxidase (HRP)‐conjugated secondary antibodies (Dako North America Inc.) were used to stain sections after primary antibodies against ACKR3 (1:200, Proteintech) at 4°C overnight. Following the manufacturer's instructions, the DAB Color Development Kit (DAKO) was used to measure antibody expression. The nuclei were counterstained with hematoxylin before the slides were photographed and examined using an optical microscope (Olympus Co.).