Eclipse ts100 inverted

Following euthanasia, livers were fixed in 10% formalin (Thermo Fisher Scientific) in Sodium Chloride (NaCl), 0.9% (w/v) Aqueous, Isotonic Saline (RICCA Chemical) overnight at 4 °C. Tissue was then incubated in 15 and 30% sucrose gradients overnight each diluted in 0.9% (w/v) NaCl, embedded in optical cutting temperature compound (Tissue Tek), and stored at −80 °C. Liver lobules were cryosectioned (6 µm) in the transverse axis, and sections were washed with 0.1% Triton X-100 in phosphate-buffered saline (PBS) solution (PBS-T) and immersed in mounting medium with the nuclear fluorescent dye 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Vector laboratories). Slides were immediately visualized using fluorescent filters for DAPI and the Rainbow colors (mCerulean, mOrange, mCherry and CAG-EGFP). Images were acquired with the Nikon microscope (Eclipse 80i upright fluorescent or Eclipse TS100 inverted). For image processing, analysis and cell counting, Adobe Photoshop and Image J (Fiji) software were used.

Cell migration was determined using the wound healing assay. Equal number A549 or PC-3 cells (4.0 × 10⁵/ml) were seeded in 6-well plates and incubated at 37°C in 5% CO₂ for 24 h in growth media with 10% FBS (Kansas, USA) media to allow cells to attach onto the plate to form a monolayer. The growth media was changed to serum-free medium containing of Mitomycin-C (Calbiochem, USA) at 1.0 μg/ml and further incubated in 37°C for 2 h to inhibit cell proliferation, before wounds of similar size were introduced into the monolayer by a sterile pipette tip. Cell debris generated from the scratch were washed with 1x PBS twice, and treated with ACA stand alone, rhAFP stand alone, or combination of rhAFP and ACA in serum-free medium for 24 h at 37°C. The images and speed of wound closure was documented at 0 h and 24 h post-wounding using the Nikon Eclipse TS100 inverted fluorescence microscope (Nikon Instruments, Japan) and analyzed using TScratch software, Version 1.0 (MathWorks Inc., USA).

Cell invasion capacity of selected sub-cell lines were examined using transwell invasion assay by measuring the number of cells transmigrating through a layer of extracellular matrix, Matrigel. Equal number A549 or PC-3 cells (1.0 × 10⁵/ml) were seeded in 6-well plates and were treated with ACA stand alone, rhAFP stand alone, or combination of rhAFP and ACA for 24 h at 37°C. 24-well transparent PET membrane 8.0 μm pore size insets were coated with 70.0 ul of 1.5 mg/ml Matrigel (BD Biosciences, USA). Cells were starved with serum free media and harvested after 20 h. Cells were resuspended with 500.0 μm of serum free media and were added to the upper insert, whilst media with 20.0% (v/v) FBS was added at the lower insert as a chemo-attractant. The cells were incubated for 24h at 37^o C. Cells in the upper insert were removed with cotton swabs, and invading cells on the underside of the membrane were fixed in 100.0% ethanol for 2 min, followed by staining with 1.0% (w/v) methylene blue (Sigma, USA) for 20 min. Number of invaded cells in eight random fields of each transwell invasion membrane insert area were counted under the Nikon Eclipse TS100 inverted microscope (Nikon, Japan) at 200× magnification.