Conductive indium tin oxide glass slides

For MALDI-MSI, sections were collected on conductive indium-tin-oxide glass slides (Bruker Daltonics, Bremen, Germany). Muscle sections were subjected to washing steps using 70% and 95% Ethanol to deplete lipids, and were dried in a desiccator for 30 min. The matrix was applied using the ImagePrep station (Bruker Daltonics) according to the protocol detailed in Table 1. The matrix was 10 mg/mL sinapinic acid in water/acetonitrile 60:40 (v/v) with 0.2% trifluoroacetic acid.
The MALDI spectra were acquired on an Autoflex Speed MALDI-TOF/TOF mass spectrometer with a Smartbeam laser using FlexControl 3.4 and FlexImaging 3.0 software packages (Bruker Daltonics). For protein imaging, ions were detected in positive linear mode at a mass range of m/z 2000–20,000 with a sampling rate of 0.63 GS/s. The lateral resolution was set to 100 μm and a total of 500 laser shots were accumulated per pixel at constant laser power. Deflection was set at m/z of 1500, and laser focus at medium. Analysis were performed using a detector gain of 2.783 V, ion source voltage 1 at 19.5 kV, ion source voltage 2 at 18.15 kV and lens voltage at 7 kV. A protein standard (Bruker Daltonics, Bremen, Germany) was employed for external calibration of spectra, which was done externally on the same target before each measurement.

Flash frozen tissues were cut (14 μm) using a cryostat-microtome (Leica CM3050S; Leica Microsystems, Welzlar, Germany), and thaw-mounted onto conductive indium tin oxide glass slides (Bruker Daltonics, Bremen, Germany). Sections were dried under a flow of nitrogen and desiccated at room temperature for 15 min, coated with 2,5-dihydroxybenzoic acid (35 mg/ml in 50% AcN, 0.2% TFA) before analysis using MALDI-TOF/TOF (Ultraflextreme, Bruker Daltonics). The Smartbeam II 2 kHz laser was operated in positive ion mode. Purified α-1, α-2 and β-1 were spotted on one section as an in-situ reference.