Cm1100 cryostat

Twenty-micrometer thick sections were cut from the frozen scent glands of muskrats with an optimal cutting temperature compound (OCT, Sakura Finetek USA, Inc., Torrance, CA, USA) using a Leica CM1100 cryostat (Leica Microsystems, Inc., Wetzlar, Germany) and collected onto the indium–tin oxide-coated microscope glass slides (ITO slides, 80 Ω, 2.5 × 7.5 cm, Bruker Daltonics, Berman, Germany). Before the matrix coating, the tissue sections were dried at room temperature and captured on an Epson Perfection V550 Photo Scanner (Seiko Epson Co., Tokyo, Japan) to obtain histological images for the correction and calibration of subsequent mass spectrometry image acquisition area.

Cryo-samples of the tumors were cut on a Leica CM1100 cryostat (Leica Microsystems, Wetzlar, Germany) into 7 µm thick slices. For each tumor, 3–4 slices were stained with hematoxylin and eosin according to the standard protocol and examined using a Leica DFC290 microscope (Leica Microsystems, Wetzlar, Germany).
The hypoxic fraction in the tumor tissue was analyzed using pimonidazole hydrochloride (Hypoxyprobe-1, Chemicon International, Temecula, CA, USA). Hypoxyprobe-1 was administered to mice intraperitoneally at a dose of 60 mg/kg 90 min prior to sacrification. Cryosections were stained with IgG1 mouse monoclonal antibodies conjugated with FITC at a dilution of 1:200 at 37 °C in a wet chamber for 3 h. The obtained tumor slices were examined on a Leica DMIL 31 LED fluorescence microscope (Leica Microsystems, Wetzlar, Germany), fluorescence was excited at 488 nm, and a signal was recorded in the range of 500–540 nm. The relative hypoxic fraction was calculated in the ImageJ software (NIH, Bethesda, MY, USA) as a percentage of the pimonidazole-positive area of the total tumor area.

Tumor sample was embedded in Tissue-Tek OCT (Optimum Cutting Temperature; Sakura, Berkshire, UK), by freezing in liquid nitrogen-cooled 2-methyl butane (Sigma-Aldrich, Dorset, UK) both prior to and following microfluidic culture. Tissue sections of 8 µM were cut on a Leica CM1100 Cryostat and fixed for 20 min in -20°C cooled methanol, stained in Harris’ Hematoxylin (Sigma-Aldrich) for 5 min before differentiating in 1% acid alcohol (concentrated HCl in 70% Ethanol) and rinsing in running tap water. The tissue was then stained with 1% (w/v) Eosin Y (Sigma-Aldrich) in tap water for 5 min before washing in tap water. The tissue was dehydrated through graded alcohols (70, 90 and 100%) and three changes of histoclear before mounting in histomount. Slides were visualized using light microscopy (Nikon ECLIPSE E800) and photographed (Micropublisher 5.0 Real Time Viewing) utilizing Image-Pro premier software (MediaCybernetics, Cambridge, UK). Detailed observations of the tissue characteristics were carried out under close supervision of a consultant head and neck pathologist, Lazslo Karsai.