Hematoxylin eosin solution

The ear and back skin tissues of the mice were removed, fixed with 10% formaldehyde, embedded in paraffin, and serially sectioned into 4–6-μm sections using a microtome (Leica Biosystems, Newcastle, United Kingdom). The tissues were mounted on slides and stained with hematoxylin/eosin solution (Sigma-Aldrich) or toluidine blue (Toluidine Blue Stain Kit, VitroVivo Biotech, Rockville, MA, United States). For immunohistochemical staining, the slide-mounted tissues were incubated overnight at 4°C with CD3 primary antibodies (1:150 dilution; cat.no. ab16669, Abcam, Cambridge, United Kingdom) and processed using a XT System Benchmark autostainer (Ventana Medical System, Tucson, AZ, United States) according to the manufacturer’s instruction. The cells positive for CD3 and toluidine blue staining as measured from the tissue sections were analyzed using Solution for Automatic Bio-Image Analysis software (Ebiogen, Seoul, South Korea).

Paraffin-embedded hippocampal sections were stained with hematoxylin-eosin solution (Sigma-Aldrich) according to the manufacturer’s instructions. To assess mean microvascular density, a light microscope (Olympus, Tokyo, Japan) was applied to determine the number of microvessels per unit area (/mm²) [33 ].

After euthanizing the mice by CO₂ asphyxiation, the tumor was harvested, fixed in formalin solution (Sigma), embedded in paraffin (Sigma), sectioned (5 μm thick slices), and stained with hematoxylin-eosin solution (Sigma). Immunohistochemical staining was performed on 3-μm-thick paraffin embedded sections. Briefly, the sections were placed on SuperfrostPlus microscope slides (Fisher Scientific). Vectastain elite ABC HRP kit (Vector Labs) was used as a 3,3′-diaminobenzidine (DAB) chromogen for detecting antibodies. Sections were deparaffinized by xylene. Solution of 0.1% trypsin was applied for 10 min for antigen retrieval, followed by treatment with 0.3% H₂O₂ in methanol for 30 min. Slides were blocked by blocking buffer and subsequently incubated with primary antibodies and secondary antibodies for 30 min each. After incubating with AB and DAB reagents, the specimens were counterstained with hematoxylin-eosin and examined under light microscopy (Leica Microsystems) for histological evaluation.