Indium tin oxide slides

Adult thymus tissues of 3-month-old mdx and C57Bl mice were frozen for preparation of cryosections (thickness of 10 μm) with the use of a cryostat (CM 1900; Leica Microsystems, Wetzlar, Germany). For imaging mass spectrometry, the sections were thaw-mounted on indium–tin oxide slides (Bruker Daltonik, Bremen, Germany), dried in silica gel-containing plastic tubes and then sprayed with 9-aminoacridine (5 mg in 4 ml of 80% ethanol) with the use of a 0.2-mm nozzle calibre airbrush (Procon Boy FWA Platinum; Mr Hobby, Tokyo, Japan) for matrix-assisted laser desorption–ionization (MALDI) imaging mass spectrometry in negative-ion mode. Adjacent sections were stained with H&E. Imaging mass spectrometry was performed with iMScope TRIO Mass Microscope (Shimadzu, Kyoto, Japan). MALDI mass spectra were acquired with a laser diameter of 50 μm, 200 shots/spot, scanning pitch of 20 μm, and scanning m/z range of 615–931. Regions of tissue samples exposed to the laser radiation were determined by light and fluorescence microscopic observations.

TMAs from formalin-fixed, paraffin-embedded tissue from patients diagnosed with exocrine pancreatic cancer were prepared at the Institute of Pathology, Charité—Medical University Berlin. For MALDI imaging, a 6-µm section from a paraffin block was prepared on the microtome and transferred to indium tin oxide slides (Bruker Daltonik, Bremen, Germany) by decreasing concentrations of ethanol (modified after Caprioli et al.) [5 (link)] and antigen recovery was performed (modified after Gustafsson et al.) [16 (link)]. An automatic spayer was used to apply Trypsin and matrix solutions (α-cyano-4-hydroxycinnamic acid, (HTX Sprayer). In total 550 µL trypsin solution (20 µg, 20 mM ammonium bicarbonate) was applied to the section. After incubating the tissue (2 h at 50 °C; humid chamber), the matrix solution (1 mL 7 g/L α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 1% trifluoroacetic acid) was applied also with the HTX sprayer (75 °C, estimation cycle 1.80).

After obtaining the blood, the hearts of three rats each from the H, S, and MOD groups were quickly separated, excised, snap-frozen in liquid nitrogen and stored at −80°C for MALDI–MSI.
The snap-frozen hearts were fixed to the sample holder using ultrapure water, and 10-µm-thick transverse slices were prepared using a cryostat microtome (Scotsman Jencons, Germany) at −17°C. The distance from the slice position to the apex was 2/5^th of the vertical length of the heart. The tissue slices were then transferred onto indium tin oxide slides (Bruker Daltonics, Germany) and dried in a vacuum pump for 30 min. Matrix spraying and MALDI–MSI were then conducted, as previously described (Liu et al., 2014 (link); Liu et al., 2017 (link)). Molecular spectrum peaks were selected in the MALDI–MSI results, and statistical analysis was performed using the SCiLS Lab software based on the normalization of total ion chromatography.