Cm 1950 uv cryostat

For CAL samples, a female NGS untreated mouse aged 10 weeks and weighing 25 g was euthanized and the brain was dissected and snap-frozen in liquid nitrogen. Frozen brain was divided longitudinally in two hemispheres. From the whole two hemispheres, 10 µm-thick serial sections were made using a Leica CM 1950 UV cryostat and stored at −80 °C before sample preparation. All killings for organ removal were performed according to German Laws for Animal Protection and approved by the Institutional Review Board and the responsible animal welfare officer of the Deutsches Krebsforschungszentrum (DKFZ) (internal reference number DKFZ374).

Fresh frozen human lung cancerous tissue sectioning was performed on a LEICA CM1950UV cryostat to obtain sections of 14 µm thickness, and were thaw-mounted on Indium Tin Oxide (ITO)-coated glass slides (Bruker Daltonik GmBH, Bremen, Germany). Tissue sections were treated according to Carnoy’s washing procedure (30 s in 70% EtOH, 30 s in 100% EtOH, 90 s in Carnoy’s fluid (EtOH:acetic acid:water (90:9:1 v:v:v)) and 30 s in 100% EtOH), dried for 30 min and 12 layers of 2,5-dihydroxybenzoic acid (DHB) matrix (40 mg/mL in 60/0.1 (v/v) acetonitrile/trifluoroacetic acid) was deposited on the tissue by using a SunCollect pneumatic sprayer (SunChrom, Friedrichsdorf, Germany). MALDI MSI data were acquired with a rapifleX tissue typer in single time of flight (TOF) mode (Bruker Daltonik GmBH, Bremen, Germany), as described earlier [23 (link)]. The resulting mass spectra will be further processed in R software (Cardinal) [52 (link)].

Liver and kidney tissues from mice at 5 day post infection were snap-frozen in optimum cutting temperature (OCT) compound (Tissue-Tek) and sectioned serially (7 μm) using a Leica CM1950 UV cryostat. Sections fixed with cold acetone were stained with Hematoxylin/Eosin (H&E) or probed with polyclonal anti-S. aureus antibodies (abcam; ab20920, 1:100 dilution) in immunofluorescence staining using Alexafluor488-conjugated anti-rabbit IgG (Invitrogen, 1:500 dilution). DAPI was used as the counterstain (1 mg per ml, Sigma). Fluorescence images were acquired using a Leica DM4000B microscope and processed with Photoshop CC 2019 (Adobe).