Bz 8000 microscope

EC migration was analyzed by two different assays. For the scratch wound healing assay, 3.2 × 10⁵ HDMECs were seeded in 35-mm dishes and then exposed to different concentrations of linalool. After 24 h of incubation, four scratches were generated onto the cell monolayer using a 10 µL pipette tip. Immediately after scratching (0 h) and after 6 h, images of the scratches were taken using a phase-contrast microscope (Leica DFC450C;  Leica Microsystems, Wetzlar, Germany). Wound areas were measured using the LAS V4.8 software (Leica Microsystems).
For the transwell migration assay, HDMECs were seeded in 100-mm dishes and then exposed for 24 h to different concentrations of linalool. Thereafter, 2.5 × 10⁵ HDMECs treated with linalool or vehicle were suspended in 500 µL endothelial basal medium (EBM) without supplements and seeded into the inserts of 24-transwell plates (8 µm pores; Corning, Wiesbaden, Germany), while 750 µL EBM containing 1% fetal calf serum (FCS) was added to the bottom chamber. After 5 h of incubation, the non-migrated cells were removed and the migrated cells were stained with Diff-Quick (LT-SYS, Berlin, Germany) and counted under a BZ-8000 microscope (Keyence, Osaka, Japan).

After performing HCS, the reporter cells on 96-well or 24-well black culture plates were analyzed using a BZ-8000 microscope (Keyence, Osaka, Japan). Phase contrast and fluorescence images were obtained at a magnification of 20x. Fluorescence images were collected using the (Ex/Em = 360/460 nm), (Ex/Em = 470/525 nm), and (Ex/Em = 545/605 nm) filters (Keyence) for TagBFP, EGFP, and TagRFP, respectively.

Immunofluorescence analysis was performed on 3.5 μm paraffin-embedded sections from xenografts to detect tumor vessels. Paraffin-embedded sections were de-paraffinized and treated as described for immunohistochemistry. Sections were blocked with goat serum for 20 min at room temperature and incubated with rat anti-mouse CD31 antibody (1:20, Dianova, Hamburg, Germany) overnight at 4°C. The next day, the secondary antibody anti-rat IgG (H+L), F(ab’)2 Fragment (Alexa Fluorr555 Conjugate) (1:1000, CST) was applied. Sections were counter-stained with ProLong Gold Antifade Reagent with DAPI. Images were captured with a Keyence BZ-8000 microscope.

To induce tumors in vivo, Hepa1‐6 cells were injected into the spleen using a medium containing 0.2 mL medium 199 (Sigma‐Aldrich) with a concentration of 10⁷ cells/mL.¹⁸ The injection was carried out slowly after identifying the spleen. After 7 days of tumor cell injection, the mice were sacrificed, and the liver was extracted and fixed in 10% formalin overnight. Tissue sections of the liver (4 μm thickness) were stained with hematoxylin and eosin (HE) for further analysis. The relative areas occupied by the tumors were calculated as a percentage of the total scanned liver area using a BZ‐8000 microscope (Keyence).

The neurite length of βIII-tubulin-positive cells in the slices was quantified by counting the intersection of fibers with a grid of cycloids (cycloid length: 30 μm) according to a previously described stereological analytical method [26 (link)]. The length density L(A) was calculated with the formula: L(A) = 2 x (ΣQ(A))/(a(frame) x ΣP(ref)), where ΣQ(A) is the total number of intersections of fibers with the cycloids; ΣP(ref) is the total number of points associated with the test frame that fall within the reference area, and a(frame) is the area of the test frame [26 (link)]. Only the fibers that possess an ending connecting to a DAPI-positive nucleus were counted. Stereo Investigator v8.0 (Micro-Bright Field) with an Olympus BX-51 light microscope and MBF Bioscience CX9000 camera were used to define sampling areas and evaluate the cell culture frames.
The neurite length of βIII-tubulin positive cells in slices was also quantified using the Keyence BZ-8000 microscope with Keyence software according to the manufacturer’s instructions. Only the fibers extending from DAPI-positive nuclei were analyzed. For each condition, at least 80 to 100 cells were measured.