Ito coated slides

LNCaP xenograft tumour was used for the MSI studies. Tissues were cut in a way that mimics a sagittal plane of slicing, as there is no spatial orientation with xenograft tumour. Cryosectioning was performed using a Leica cryotome (CM 1850 UV, Wetzlar, GmbH & Co. KG) at −18 °C and 12 μm tissue thickness. Sections were thaw-mounted onto conductive indium-tin-oxide (ITO)-coated slides (Bruker Daltonik, Bremen, GmbH & Co. KG), dried in a vacuum desiccator at room temperature for 30 min, and then stored at −80 °C until MSI analysis.

Frozen tissues were placed at -20°C before sectioning in a Microm HM550 cryostat (Thermo Scientific™). Tissues were sectioned at 10 μm thickness and thaw mounted onto indium-tin-oxide (ITO)-coated slides (Bruker Daltonics) for MALDI MSI analysis with serial sections mounted onto glass slides for histological analyses. The microtome chamber and specimen holder were maintained between -15°C and -20°C. Slides were stored at -80°C until further processing. For desiccation experiments, slides were subjected to desiccation in a tabletop vacuum desiccator before freezing.

Parallel tissue samples (heat-fixed and untreated) from the mouse infection studies were embedded in OCT (optimal cutting temperature cryo-compound), (Sakura Finetek, Torrence, CA, USA) freezing medium, immersed in a pre-chilled isopentane bath cooled with dry ice to freeze, and then stored frozen at −80°C. Sections (10 μm) were made with a Leica cryostat (Leica CM1900 Leica Microsystems Inc. Buffalo Grove, IL, USA) and were placed on ITO coated slides (Bruker Daltonics, Billerica, MA, USA) for MALDI-MSI. Serial sections (5 μm) were hematoxylin and eosin (H&E) stained for morphological analysis. After sectioning, the MALDI-MSI slides were rinsed in 70% ethanol for 30 seconds, followed by 95% ethanol for 30 seconds and a brief water wash to remove the OCT compound. Finally, the slides were again rinsed in 70% ethanol and 95% ethanol to dehydrate the tissue. After 1 hour in a dessicator, slides were sprayed with sinapinic acid (SA: 10 mg/ml in 50% acetonitrile, 1% TFA) for protein detection or 2,5-dihydroxybenzoic acid (DHB: 30 mg/ml in 50% methanol, 1% TFA) for lipid detection, in an ImagePrep spraying device (Bruker Daltonics) using the manufacturer’s recommended method for SA or DHB application respectively. After matrix application, the slides were stored in a dessicator under vacuum for 1 hour before MALDI-MSI.