Anti adh6

For immunohistochemical (IHC) staining, tissues were embedded in paraffin after fixed in 4% paraformaldehyde and sectioned at 4 μM. After antigen retrieval at high temperature, sections were incubated with sheep serum albumin for blocking antigen. Sections were then incubated with primary antibodies (anti-ADH1B, anti-ALDOB, anti-ADH1A, anti-FBP1, and anti-ADH6; ABclonal Technology) for overnight. Secondary antibody was applied for 30 min. After added the diaminobenzidine solution, sections were stained with hematoxylin. The images were visualized by XSP-C204 biomicroscope.
Additionally, sections were stained with anti-mouse CD14 monoclonal antibody (Proteintech, Wuhan, China) for 15 h, then incubated with FITC-conjugated goat anti-mouse IgG (Proteintech) for 1 h. After washing with solution for 4 times, sections were incubated with sheep serum albumin for blocking antigen. Then sections were stained with anti-rat FOXP3 polyclonal antibody (Absin, Shanghai, China) for 15 h, then incubated with Cy3-conjugated goat anti-rat IgG (Proteintech) for 1 h. Nuclei were counterstained with DAPI. The images were visualized by XSP-C204 biomicroscope.

Tumors tissues and normal tissues samples were homogenated in RIPA lysis buffer with PMSF on ice. Proteins were extracted and then quantified with BCA protein assay kit (Beyotime, Shanghai, China). Then 20 µg proteins were separated on SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% skim milk at room temperature for 2 h, membranes were incubated with primary antibodies (anti-ADH1B, anti-ALDOB, anti-ADH1A, anti-FBP1, anti-ADH6, and anti-β-actin; ABclonal Technology, Wuhan, China) at 4°C for overnight, respectively. Subsequently, membranes were incubated with HRP-linked secondary antibodies and detected with an ECL chemiluminescence kit (Beyotime). Quantification of proteins was performed by normalized to β-actin using ImageJ software.