Standard microtome

At the end of each experiment, 3/6 epidermis were fixed in a paraffin cassette and the cassettes were transferred into 70% ethanol and stored at 4°C until use. On a day of experiment, epidermis was gradually dehydrated (80% EtOH, 45 min; 90% EtOH, 45 min; 96% EtOH, 45 min, 100% EtOH, 45 min; xylene 100%, 45 min) and vertical cell membrane sections (5 µm) were cut using a standard microtome (Leica) and stained with hematoxylin-eosin stain as described by Fischer et al.24 (link) Sections were examined microscopically to evaluate any eventual cell membrane or intracellular changes.

The entire mouse liver was harvested, weighed and rinsed with cold phosphate buffered saline (PBS). Tumor nodules were counted, isolated and separated for total RNA and genomic DNA extraction. Samples for RNA extraction were stored in RNAlater Stabilization solution (Life Technologies) at −80 °C. RNA isolation was done using Trizol reagent (Life Technologies) according to the manufacturer's protocol. Genomic DNA was isolated using standard proteinase-K treatment, phenol-chloroform extraction and ethanol precipitation. Genomic DNA was then dissolved in sterile TE (10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8)) and quantified using a Nanodrop spectrophotometer. Histological sections were only taken for larger tumor nodules (>2 mm in diameter). Formalin fixed-paraffin embedded sections from various tissues were sectioned at 5 μm using a standard microtome (Leica), mounted and heat-fixed onto glass slides. Tissue section slides were either processed and stained with hematoxylin-eosin (HE) using standard protocols. Histopathological analyses were performed in part by board-certified human pathologists (M.A.L. and K.A.).