Hematoxylin

Mouse tumor tissues were immobilized in 4% paraformaldehyde, embedded in paraffin and, cut into 4 μm sections. After deparaffinization, permeabilization, antigen retrieval, and incubation in 3% H₂O₂, tissue sections were incubated with primary antibodies PCNA (1:500, Abcam), PKM2 (1:800; Cell Signaling Technology), PKM1 (1:1000; Cell Signaling Technology), hnRNPA1 (1:100; Abcam). Then, the sections were probed with goat anti-rabbit biotinylated secondary antibody (1:3000, Abcam). Immunoreactive signals were visualized using DAB HRP Kit (Beyotime Biotechnology, Shanghai, China) and the nuclei were counterstained with hematoxylin (Sigma Aldrich).
hematoxylin and eosin (HE) was performed to stain paraffin-embedded tissue sections. Sections were cut into 4 μm slices, and cytoplasm and nuclei were stained with eosin (Sigma Aldrich) and hematoxylin. The images were observed under a microscope.

The tumor tissues were fixed with 4% formaldehyde at 4 ˚C overnight and then embedded with paraffin. The tissues were sectioned into 4–5 µm slices. For hematoxylin and eosin (HE) staining, the dewaxed sections were stained with hematoxylin (Sigma, St. Louis, MO, USA) for 15 Min, and then incubated in hydrochloric acid alcohol (1%) for 15 Sec. After soaking in eosin (Sigma) for 2 Min, the slices were observed under an optical microscope. For theimmunohistochemical (IHC) assay, the sections were stained with F4/80 macrophage marker, EZH2, CCL5, or MMP2 (1:100, ab100790; Abcam, USA). In brief, after deparaffinized with xylene, the sections were subject to retrieval in a heating citrate buffer (pH 6.0) for 15 Min. After cooling down to temperature, the sections were blocked with normal goat serum (Thermofisher) at 37 ˚C for 10 Min and then incubated with primary anti‐F4/80, EZH2, CCL5, or MMP2 antibodies overnight at 4 °C. Next, sections were incubated with anti‐rabbit secondary antibody (biotinylated) (1:1,000, ab6720, Abcam) at room temperature for 30 Min. The horseradish peroxidase‐conjugated streptavidin (Amersham, Amersham, UK) was incubated with sections at 37 ˚C for 30 Min. The diaminobenzidine (Sigma) was used for chromogenic detection. The staining was observed under an inverted microscope (Olympus, Tokyo, Japan) (400× magnification).

Six to nine 8.0 μm sections were mounted on uncoated glass slides and stored at −80°C until processed further. For each specimen, one slide was stained with Hematoxylin (Sigma Aldrich, St. Louis, MO) and Eosin (Sigma Aldrich, St. Louis, MO) and examined by a certified pathologist (LL) to confirm the presence of malignant cells. LCM was used to isolate tumor epithelial cells from the surrounding microenvironment as previously described [49 (link)]. Briefly, slides were fixed in 70% ethanol, rinsed with deionized water, stained with Hematoxylin (Sigma Aldrich, St. Louis, MO) and Scott's Tap Water (Electron Microscopy Sciences, Hatfield, PA), and dehydrated in ethanol (70%, 95% and 100%) and xylene. Complete mini protease inhibitors (Roche Applied Science, Indianapolis, IN) were added to the 70% ethanol, deionized water, Hematoxylin, and Scott's Tap Water to preserve protein and phosphorylations from degradation.
Using the infrared laser of a Veritas microdissection system (Arcturus Bioscience, Mountain View, CA) 0.5-18 mm² of malignant cells were collected from each sample on CapSure Macro LCM caps (Arcturus Bioscience, Mountain View, CA).