Unlocking Lipid Spatialomics in FFPE Tissues via MALDI Mass Spectrometry Imaging : Lipid MSI raw files of Rat brain images with DHB matrix

Tissue Samples and Experimental Design For method development, two male Wistar rats (n = 2) were used. Rat brains were obtained through a collaboration with Dr. Dasa Čížková (Institute of Neuroimmunology, Slovak Academy of Sciences, Bratislava). All animal procedures were conducted in accordance with institutional animal-care guidelines and international standards and were approved by the State Veterinary and Food Committee of the Slovak Republic (Ro-4081/17-221) and the Ethics Committee of the Institute of Neuroimmunology. Adult male Wistar rats were sacrificed by CO₂ asphyxiation and brains were rapidly dissected. For fresh-frozen (FF) samples, brain tissues were immediately snap-frozen in isopentane cooled to −50 °C and stored at −80 °C; FF sections were cut at 8 µm (sagittal) on a cryostat at −20 °C, mounted on ITO-coated slides and stored at −80 °C. Six FF sections were prepared. For FFPE blocks, freshly dissected brains were immersed in 10% neutral-buffered formalin (4% formaldehyde, pH 7.0; fixative-to-tissue ratio ≥ 10:1) for 12–48 h at room temperature, dehydrated through graded ethanol (70, 80, 95, 100%), cleared in xylene (2–3 changes, 30–60 min each), infiltrated with paraffin at ≈60 °C and embedded; blocks were stored at 4 °C. FFPE sections were cut at 8 µm (sagittal) on a microtome at room temperature in a single session. Twenty-four consecutive sections were prepared, of which 12 were mounted on water-treated slides and 12 on slides coated with 2% ovalbumin. In addition, four consecutive FFPE sections from each of three clinical cancer biopsy blocks (oropharyngeal carcinoma, breast carcinoma and glioblastoma) were mounted on ITO-coated slides using 2% ovalbumin as an adhesive coating. Glioblastoma tissue was obtained from the Pathology department of Lille Hospital, France, from a prospective cohort of 50 patients enrolled between 2014 and 2018 (NCT02473484), approved by the Lille Hospital ethics committee (ID-RCB 2014-A00185-42) in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. A single glioblastoma sample from this cohort was used for the present study, independently of the cohort's original objectives. Breast carcinoma tissue was obtained from Centre Oscar Lambret (Lille, France) from sixteen patients selected by expert breast pathologists. The study complied with the Declaration of Helsinki and Good Clinical Practice, approved by the Lille Hospital ethics committee (ID-RCB 2014-A00185-42) and the CPP Ile de France II (project no. 21.04.21.68809 RIPH2 HPS); written informed consent was obtained from all patients, and samples were anonymized before analysis. A single breast carcinoma sample from this cohort was used for the present study, independently of the cohort's original objectives. Oropharyngeal carcinoma tissue was obtained from Gustave Roussy (Villejuif, France). Insert ethics committee approval reference and patient-consent statement. A single oropharyngeal carcinoma sample from this cohort was used for the present study, independently of the cohort's original objectives. Deparaffinization Conditions FF tissues were dried in a desiccator prior to matrix deposition. FFPE slides were subjected to one of four deparaffinization conditions: (1) no treatment; (2) paraffin removal only (two xylene baths, 5 min each); (3) deparaffinization and rehydration (two xylene baths of 5 min, then graded ethanol [2 × 90%, 1 × 30%] and two baths of 10 mM NH₄HCO₃, 5 min each); and (4) deparaffinization, rehydration and antigen retrieval (as Condition 3, followed by 20 mM Tris buffer, pH 9, at 90 °C for 20 min, two 1-min rinses in 10 mM NH₄HCO₃, and vacuum drying). Matrix Deposition for Lipid MSI Norharmane and DHB were applied with the HTX M5-Sprayer or by sublimation11. Norharmane served as matrix for both positive and negative lipid imaging; DHB was used in positive mode only. Norharmane (spray): 7 mg/mL in CHCl₃/MeOH (2:1, v/v); 30 °C, 10 psi, CC pattern, 0.1 mL/min, 1200 mm/min, 12 passes, 2 mm track spacing. Norharmane (sublimation): 20 mg in 1.5 mL MeOH, ≈5 min. DHB (spray): 10 mg/mL in MeOH/0.1% TFA (7:3, v/v); 75 °C nozzle, 55 °C tray, 10 psi, CC pattern, 0.1 mL/min, 1200 mm/min, 8 passes, 2 mm track spacing. DHB (sublimation): 20 mg in 1.5 mL acetone, ≈5 min. Tissue Preparation for Peptide MSI Serial sections were deparaffinized (two xylene baths, 5 min), rehydrated (graded ethanol 2 × 90%, 1 × 30%; two 10 mM NH₄HCO₃ baths) and subjected to antigen retrieval (20 mM Tris, pH 9, 20 min at 90 °C; two 1-min 10 mM NH₄HCO₃ rinses; vacuum drying). Trypsin (40 µg/mL in 50 mM NH₄HCO₃) was applied with the HTX M5-Sprayer; slides were incubated overnight at 56 °C in a MeOH/H₂O-saturated chamber and vacuum-dried. HCCA-aniline matrix (10 mg/mL in 70% ACN / 0.1% TFA with 0.72% aniline) was sprayed in gradual layers and dried before analysis. MALDI MSI Acquisition Lipid and peptide images were acquired on a timsTOF fleX (Bruker Daltonics, Bremen, Germany) in MALDI mode with a SmartBeam 3D laser, in delayed-extraction reflectron mode, 300 laser shots/pixel, 60 µm spatial resolution. Lipids: m/z 400–1350, positive and negative ion modes, ≈85% laser energy. Peptides: m/z 320–3200, positive ion mode, ≈80% laser energy. Source voltages were 20 kV and 11 kV (lens). Imaging data were processed in SCiLS Lab.