Xylene substitute

All the standard molecules used in the study were purchased from Sigma-Aldrich (St. Louis, MO, USA): morphine, morphine-3-glucuronide, morphine-6-glucoronide solutions (1.0 mg/mL in methanol each standard) and 6-acetylmorphine (1.0 mg/mL in acetonitrile standard solution). Deuterated internal standards (ISs) were purchased from Sigma-Aldrich as well: morphine-D₃ (1.0 mg/mL in methanol standard solution), 6-acethylmorphine-D₃ (1.0 mg/mL in acetonitrile), morphine-3-glucuronide-D₃ (100 µg/mL in methanol with 0.05% of NaOH) and morphine-6-glucuronide-D₃ (100 µg/mL methanol/water (1:1, v/v)). Solvents used in the extractive processes were purchased from Sigma-Aldrich (methanol, hydrochloric acid, chloroform, dichloromethane and ethyl acetate) and VWR Chemicals (Radnor, PA, USA) (acetone, acetonitrile, isopropanol and n-hexane). Buffer pH 6.88 solution was purchased from PanReac (Barcelona, Spain). For histological inclusions, absolute ethanol and xylene substitute were purchased from VWR Chemicals and high fusion point paraffin pearls Histowax® were purchased from International Medical Products (Brussel, Belgium).

FFPE samples were prepared by cutting multiple 20 μm scrolls of the tissue and dewaxing the tissue in a xylene substitute (VWR, Radnor, PA, USA, 89370-090) followed by ethanol rehydration steps going from 100% ethanol to 100% DI water. The DNA extractions were performed with various starting amounts of tumor tissue ranging from 5 mg to 50 mg. Tissues were weighed after deparaffinization of FFPE scrolls or thawing cryopreserved tissue using the Denver Instrument TP-114. DNA was extracted using an Extracta DNA buffer (Quantabio, Beverly, MA, USA, 95091) combined with proteinase K (Zymo, Dustin, CA, USA, D3001-2) at a final concentration of 1 mg/mL. Tissues were incubated in a cell lysis solution at 55 °C for 3 h in an Incu-shaker Mini (H1001-M). DNA was purified using the Zymo Research DNA Clean and Concentrator-5 Kit (11-302C). The samples were purified to Zymo’s specifications, except 10 μL of DNA Elution Buffer was used in place of the procedural 6 μL. Each centrifugation step was performed using the Eppendorf Centrifuge 5425R set at 21 °C and 14,000 RPM for 60 s.

Pellets were fixed in 10% (v/v) formalin (Anachemia) overnight at 4°C, washed in PBS, processed, and embedded in paraffin wax. Embedded pellets were cut at 5-μm thickness using a Leica RM2125 RTS rotary microtome (Leica Biosystems). Sections were deparaffinized using Xylene Substitute (VWR International) and stained with 0.1% (w/v) Safranin-O (Sigma). Sections were then counter-stained with 1% (w/v) Fast Green FCF (Sigma) and mounted in Richard-Allan Scientific Mounting Medium (Thermo Fisher). The resulting stained sections were imaged on a Nikon Eclipse Ti-S microscope coupled to a DS-U3/Fi2 Color CCD camera using 10x and 20x objective lenses.
For calcium staining, sections were deparaffinized, as described above. Sections were stained with Alizarin Red S (Sigma–Aldrich; A5533) solution [2% (w/v), pH 4.2] for 5 min. Sections were then dehydrated in acetone and acetone: xylene 1:1 solution. Finally, sections were cleared in UltraClear Xylene Substitute (VWR International), mounted, and imaged as described above.