Balkan Journal of Medical Genetics

RENOPATHOLOGICAL MICROSTRUCTURE VISUALIZATION FROM FORMALIN FIXED KIDNEY TISSUE BY MATRIXASSISTED LASER/DESORPTION IONIZATION-TIME-OFFLIGHT MASS SPECTROMETRY IMAGING
Fröhlich S1, Putz B1, Schachner H2, Kerjaschki D2,Allmaier G1, Marchetti-Deschmann M1,*
*Corresponding Author: Dr. Martina Marchetti-Deschmann, Vienna University of Technology, Institute of Chemical Technologies and Analytics, Getreidemarkt 9/164-IAC, 1060 Vienna, Austria; Tel.: +43-1- 58801-15152; Mobile: +43+664-605887663; Fax: +43-1-58801-915162; E-mail: martina.marchettideschmann@ tuwien.ac.at
page: 13

MATERIALS AND METHODS

Chemicals were of analytical grade (Sigma- Aldrich, St. Louis, MO, USA). Water (ddH2O) was purified in a Simplicity system (Millipore, Billerica, MA, USA). Frozen rat kidney tissue (formalinfixed, unfixed) embedded in paraffin (FFPE), optimal cutting temperature (OCT) compound (Sakura Finetek, Tokyo, Japan) or sucrose was sliced to 10, 5 and 0.5 mm using a cryo-microtome (Leica, Wetzlar, Germany) and mounted on indium-tin oxide coated glass slides (Sigma-Aldrich). The OCT compound and sucrose were removed by washing the samples five times (4°C) for 45 seconds with 200 mL ddH2O/cm2. A xylene bath followed by a descending ethanol gradient (100.0, 96.0, 70.0, 50.0, 0.0% in ddH2O) removed the paraffin. All samples were washed three times with 200 mL 70.0% ethanol/ cm2 for 45 seconds (4°C) and vacuum dried for 15 min. before the MALDI matrix application. Samples were hematoxylin/eosin (H/E) stained before MSI treatment. The MALDI matrices [a-cyano-4-hydroxycinnamic acid (HCCA), sinapinic acid, 2,5-dihydroxybenzoic acid] were dissolved in solvents containing 50.0 or 70.0% acetonitrile or ethanol in 0.1% aqueous trifluoroacetic acid. Matrix deposition was performed using a ChIP-1000 (Shimadzu Biotech Kratos Analytical). For protein identification, tissue was trypsinized before matrix deposition by depositing depositing enzyme directly on pre defined tissue spots [1 ng/mL in 50 mM ammonium bicarbonate, 0.1% Rapigest (Waters, Manchester, Greater Manchester, UK)]. Samples were incubated in a humidified atmosphere overnight at 37°C before desiccation in vacuum and heat treatment (85°C). Mass spectrometry imaging experiments were performed using a MALDI-TOF (time of flight) instrument (AXIMA TOF2; Shimadzu Biotech Kratos Analytical, Manchester, Greater Manchester, UK; 337nm nitrogen laser, 20Hz) and a MALDI-QqTOF instrument (Synapt HDMS, Waters; 355nm Nd- YAG laser, 200Hz). The optical diameter of 60 mm was reduced to an operational resolution of 35 mm by over sampling. Peptide and lipid identification was based on post source decay (PSD) and collision induced dissociation (CID) fragmentation in combination with database search [proteins: SwissProt (http:// www.uniprot.org/help/ uniprotkb); lipids: Lipid Maps (http://www.lipidmaps.org/ data/structure/)]. Selected ion images were generated using BioMAP (Novartis, Basel, Switzerland).

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