Cm1860 uv cryotome

Tissue sectioning (12 μm,
at −20 °C) was performed on fresh-frozen tissues using
a Leica CM1860 UV cryotome (Wetzlar, Germany). Slides with tissue
sections and cells were handled according to the same protocol: samples
were kept at −80 °C prior to analysis. Beforemass spectrometry
imaging (MSI), sublimation of 80 mg of norharmane at 140 °C for
180 s was performed using an HTX sublimator (HTX Technologies, USA).
The sample preparation of tumor xenografts was the same for offline
and online recognition.

The liver, lungs, and tumors were immediately fixed in 4% neutral-buffered formaldehyde or Bouin’s solution (Sigma-Aldrich). The tissue was then dehydrated in a gradient of ethanol concentrations and embedded in paraffin. Sections of 5 µm were stained with hematoxylin/eosin or Gomori trichrome to visualize small tumors and metastasizing B16F10 cells. For immunohistochemical staining of CD8+ T lymphocytes and NK cells within tumors, the formaldehyde-fixed samples were processed through a saccharose gradient, embedded in a freezing medium (Tissue Freezing Medium, Leica Biosystems), frozen at -80°C and cut into 10 µm sections on a Leica CM 1860 UV cryotome. Afterward, antigen retrieval was performed on the sections, which were boiled in a sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) in a microwave oven at 750 W for 6 minutes. The sections were then incubated overnight at 4°C with either anti-CD8 alpha antibody diluted 1:200 (Abcam, ab237723) or anti-NK1.1 antibody diluted 1:100 (Abcam, ab289542) and visualized by fluorescently-labelled secondary antibodies. The slides were then mounted with 15 µl of Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories), upon which they were examined and photographed using a fluorescent microscope (Olympus BX51 with an Olympus DP-2 camera).