Dp25 camera

Tissues were fixed in Excel fixative (American Master Tech Scientific, Lodi, CA, USA) and processed with paraffin-embedding procedures. Sections (5 μm in thickness) were cut and stained with hematoxylin and eosin, and the stained sections were observed under a light microscope (Olympus BX41, Center Valley, PA, USA) to assess extravasation of blood cells. Images were captured with an Olympus DP25 camera.

For immunocytochemistry, five days after seeding, cells were scraped in cold PBS, formalin-fixed, paraffin-embedded, and processed as below. For immunohistochemistry, 4 μM sections of formalin-fixed, paraffin-embedded specimens were heated at 60°C, deparaffinized in xylene, and hydrated in a series of ethanol dilutions. Epitope retrieval was by microwaving (5 min at 850w, 15 min at 150w) in 10 mM Tris-EDTA buffer pH 9.0. Slides were blocked 10 minutes in 3% BSA in TBST (Tris pH 7.6, 0.05% Tween-20). Primary antibodies were as follows: MITF, 1:100 in 3% BSA in TBST, clone D5 (Dako M3621); AXL, 1:100 in 3% BSA in TBST, clone C89E7 (CST 8661). Slides underwent 10 min peroxidase block in 3% H2O2. Secondary antibodies were: goat anti-mouse IgG-HRP (BioRad 170-6516, 1:200 in 3% BSA in TBST; Dako EnVision anti-rabbit (K4003, ready-to-use). Slides were developed with DAB+ (Dako K3468) for 10 min and counterstained 1 min with hematoxylin (Vector H-3401) prior to dehydration and mounting. Slides were imaged on an Olympus BX51 microscope with Olympus DP25 camera using Olympus WHN10X-H/22 oculars, Olympus UPlan FL N -20×/0.50 and -40×/0.50 objectives, an Olympus DP25 camera, and images acquired using Olympus DP2-TWAIN software and Adobe Photoshop 7.0. Slides were scored for intensity and distribution of AXL and MITF by a dermatopathologist blinded to clinical outcome.

Approximately 30,000 cells/well were plated on a four-well glass chamber slide (Millipore). Cells were washed in PBS 24 hours later and treated with vehicle, cetuximab (100nM) and/or honokiol (20uM). Again, 24 hours later cells were washed with PBS again. Cells were treated with 3uM MitoSox in HBSS for 30 minutes. Cells were washed in PBS and then fixed at room temperature with 4% methanol-free formaldehyde for 15 minutes. Cell were washed with PBS and treated with DAPI (Molecular Probes) for 5 minutes. Slides were mounted with Prolong gold mounting solution (Life Technologies). An Olympus BX41 microscope and Olympus DP25 camera was used for fluorescent microscopy and photography. Quantified average cell fluorescence of ~20 cells per treatment arm using ImageJ analysis.