G23301

The immunohistochemistry of MPO was conducted using mouse anti-MPO antibodies (Servicebio, Wuhan, GB12224) and HRP-labeled goat anti-mouse antibodies (Servicebio, Wuhan, G23301) along with DAB chromogenic agent kit for histochemistry (Servicebio, Wuhan, GB12224). All procedures were carried out on the basis of the manufacturer’s instructions. After blocking the fixed tissue with serum, we added primary antibodies, secondary antibodies, and developer before staining the nuclei with DAPI. Afterwards, images of the tissue under the microscope were taken and analyzed. Hematoxylin-stained nuclei appeared blue, while MPO expression was marked by a brownish yellow color.

Myeloperoxidase (MPO) immunohistochemistry was performed using mouse anti‐MPO (Servicebio, GB12224) and HRP‐labelled goat anti‐mpO (Servicebio, G23301) antibodies and the histochemical DAB chromogenic agent kit (Servicebio, GB12224). All procedures were performed based on manufacturer's instructions. The fixed tissue was first blocked with serum. Subsequently, the primary antibody, secondary antibody and developer were added. Finally, the nuclei were stained with DAPI. Then, the tissue was examined under a microscope, and images were captured and analysed. The haematoxylin‐stained nuclei appeared blue, whereas positive mPO expression was indicated by a brownish yellow colour.

Twenty ESCC patients who provided cancer and paracancerous tissue samples were randomly selected for in-patient and outpatient examination in our hospital. All tissues were confirmed by senior pathologists. Paraffin-embedded sections of selected ESCC and adjacent normal tissues were stained with HE and used for tissue and cell morphological observations. Gas6 and Axl expression were examined using immunohistochemistry. First, paraffin sections were dehydrated, antigen repair was performed, endogenous peroxidases were quenched, and serum was used for blocking. Tissues were then incubated with anti-GAS6 primary antibody (67202, Cell Signaling Technology (CST)) and secondary antibody (G23301, Servicebio), nuclei were counterstained with DAPI, and tissues were mounted on slides and dehydrated. The intelligent high-content cell imaging analysis system Invitrogen EVOS FL Auto2 (Thermo Fisher Scientific, Waltham, MA, USA) was used to observe and photograph the tissues [22 (link)]. Axl expression was detected using an immunofluorescence method [23 (link)] which involved tissue section dewaxing, antigen repair, autofluorescence quenching, incubation with primary antibody (bs-5180R, Bioss) and fluorescent secondary antibody (GB21303, Servicebio), and DAPI counterstaining of nuclei. After slides were coverslipped, EVOS FL Auto2 was used for observation and image collection.