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Preparation of Frozen Section<br />
… Preparation of Frozen Section<br />
Fresh tissues were cut about 5 mm with a razor, embedded with a compound (Surgipath FSC22: Leica Microsystems, Germany) and frozen in a −75 °C acetone bath (Histo-Tek Pino: Sakura Finetek Japan Co.,Ltd., Tokyo, Japan). The frozen samples were placed on the cryostat specimen disk and cut with the knife blade until the desired tissue is exposed. The face of the frozen sample was put on the adhesive film (Kawamoto’s film method) at 16μm thickness in a cryostat (CM3050S, Leica Microsystems, Germany). The section on the film was freeze-dried overnight at −30 °C in the cryostat (Figure S9 in the Supporting Information). For light microscopy, the frozen sections were stained with 0.05% toluidine-blue O solution for a minute and then washed with distilled water.<br /><br />
IMS Analysis<br />
The freeze-dried section on the film was attached to a glass slide (ITO coating, Bruker Daltonik GmbH) with cellophane tape. The CHCA matrix solution (7 mg/mL 80% MeOH including 0.2% TFA) was sprayed onto the glass slide using ImagePrep (Bruker Daltonik GmbH) set to the default parameters. The freeze-dried section with the matrix was analyzed in the FTICR–MS instrument using the MALDI source. The analytical conditions were as follows. MALDI control: geometry, MTP 384 ground steel; plate offset, 100.0 V; deflector plate, 200.0 V; laser power, 30.00%; laser shots, 200; frequency, 2000 Hz; laser focus, small; raster width, 30 μm. The analytical conditions of MALDI in the IMS analysis were identical to those described in the MALDI analysis. to those described in the MALDI analysis.
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