Cm1860 uv

After behavioral testing was finished, experimental rats were overdosed with tribromoethanol and decapitated. Directly prior to this, a vaginal lavage was taken from each rat to identify the day of estrous. After decapitation, the brains were removed, and flash frozen for slicing. Brains were sectioned at 40 μm using a freezing cryostat (Leica CM1860 UV) and slices were mounted on gelatin-coated slides. A cresyl violet stain was performed for verification of cannula placement under a microscope.

The organs were sectioned with a cryomicrotome (Leica CM 1860 UV) at 12-μm thickness in a 30–45% humidity atmosphere. The brain was transversally sectioned, the median lobe of the liver was axially sectioned, and consecutive transverse cross sections of the intestines (after the duodenal papillae for the duodenum) were also prepared. The tissue sections were mounted onto clean indium tin oxide (ITO) unpolished float glass slides (25 × 75 × 1.1 mm, Rs = 4–8 Ω, Delta Technologies LTD, Loveland, USA). The ITO glass slides were cleaned by sonicating for 6 min in subsequent baths of n-hexane and ethanol. Dust particles from the slides were removed using nitrogen before usage. The organs were randomly mounted onto the slides in order to minimize batch effects during analysis. Prior to analysis, the mounted tissue sections were desiccated under vacuum for 20 min. Then, 80–90 mg of norharmane (beta-carboline) MALDI matrix was dissolved in 2 ml of methanol. This solution was applied onto the slides with a sublimator device (HTX Technologies, LLC, Chapel Hill, USA) [22 ] at 140 °C for 180 s, and the samples were allowed to dry up in a vacuum desiccator for at least 20 min. Each plate was scanned (grayscale, 2400 dpi) to align the laser with a visual image. The plates were stored in vacuum bags in the − 80 °C freezer prior to MALDI-TOF analysis.