M205 stereoscope

After fixation in 10% formalin, lungs were transferred to 70% ethanol and stored overnight at room temperature. Tissues were dehydrated through a series of graded alcohols, and infiltrated with paraffin (Electron Microscopy Sciences). Tissues were embedded in paraffin and sectioned (thickness = 7 μm) on a microtome. Sections were transferred to SuperFrost Plus slides (Fisher) and allowed to dry overnight on a slide warmer (Fisher). paraffin removal was initiated by several washed in xylene, and followed by rehydration of the tissues in a series of graded alcohols. Tissues were stained in hematoxylin and eosin (H&E) solutions followed by a clearing solution of xylene. After staining, Permount^TM mounting medium (Fisher) was applied to each slide and covered with a 60 mm cover slip (Fisher). Slides were allowed to dry at room temperature overnight and then placed in a drying oven until completely dry. Images of H&E stained slides were taken using a Leica M205 Stereoscope (Leica) as well as an EVOS light microscope (Life Technologies). Macrometastases were counted under 4.32x magnification on the Leica M205 Stereoscope. Micrometastases were counted from H&E stained non-sequential sections (n = 5) from each tissue sample using an EVOS light microscope. Images were subsequently analyzed for total tumor area using ImageJ software (NIH).

All mice were handled in accordance with the Association for Assessment and Accreditation of Laboratory Animals Care international guidelines with the approval of the appropriate Institutional Animal Care and Use Committees at Indiana University, Bloomington (Protocol # 14–019–4). 2.5 × 10⁵ B16-F10 cells suspended in 100 μL phosphate buffered saline (PBS) were injected intravenously (IV) into 20 4-6 week old, female C57Bl/6 mice (Harlan) via the lateral tail vein. Mice were randomly assigned to either the control or experimental group (10 mice/group) after receiving the initial implantation of B16-F10 cells. Immediately following transplantation of melanoma cells, mice were dosed with Lunasin (30 mg / kg) or vehicle by intraperitoneal (IP) injection. The 30 mg/kg dose was selected based on our previous in vivo studies of human and mouse melanoma subcutaneous tumor growth [37 (link), 56 ]. Mice received daily IP injections of Lunasin or vehicle until the end of the experiment 18 days post-transplantation of cells; preliminary studies demonstrated that this protocol generated numerous large lesions 22 days after injection. Upon sacrificing the mice, lungs were resected and imaged using a Leica M205 Stereoscope (Leica). Tissues were fixed in 10% formalin for 72 hours and processed for subsequent histological staining.

For small interfering RNA (siRNA)-mediated knockdown of VTCN1, differentiated hESCs were transfected with either a VTCN1 human siRNA oligo duplex (Origene, SR312516, 10nM) or a control, Trilencer-27 Universal scrambled negative control siRNA duplex (Origene, SR30004, 10 nM) on BAP treatment d 3 by using Lipofectamine 2000 (Invitrogen). After 6 h, the medium was replaced with BAP medium. Total RNA was collected 24 h, 48 h and 72 h after transfection. Protein was collected 72h after transfection. At BAP treatment d 6, i.e., 72 h after the BAP 3 days, cells colonies were stained with crystal violet solution [0.2% wt/vol crystal violet in an aqueous solution containing 20% (vol/vol) ethanol and 10% (vol/vol) formaldehyde] for 15 min. After washing with water, images of individual cell colonies were captured with a Leica M205 stereoscope.