Cryostat 3050

Liver tissues from the mice were lysed in PBS and supernatants were obtained after centrifugation at 14,000 rpm for 20 min. Triglycerides concentrations from the tissue lysates were measured in milligram per decilitre (Centre facility, Department of clinical chemistry, UMG, Göttingen). The kit was used for determining the triglyceride concentration (REF: 7D74-21, http://www.ilexmedical.com/files/PDF/Triglyceride_ARC_CHEM.pdf.). The liver tissue sections (5 µm thickness, by Leica cryostat 3050) glass slides were further stained with Sudan III and H and E stains, followed by microscopy with the bright field.

Human thymic samples were fixed (for 2 hrs to overnight) in 4% PFA and processed for either cryo- or paraffin-embedding. For cryo-embedding, fixed tissue was equilibrated in sucrose 25% and embedded in O.C.T. compound (VWR). Cryosections (thickness, 7 μm) were cut on a Leica Cryostat 3050. For paraffin-embedding, a Leica PelorisII tissue processor and Sakura Tissue-Tech embedding station were used. Paraffin section (thickness, 3-5 μm) were produced using ThermoFisher rotary microtome.
Cryo- or Paraffin sections were stained with haematoxylin-eosin using an automatic station (Tissue-Tek Prisma) to verify histology of each tissue and subsequently used for immunohistochemistry analysis.

Human thymic tissues and scaffolds samples were fixed (for 2 h to overnight) in 4% PFA and processed for either cryo- or paraffin-embedding. For cryo-embedding, fixed tissue was equilibrated in sucrose 25% and embedded in O.C.T. compound (VWR). Cryosections (thickness, 7 µm) were cut on a Leica Cryostat 3050. For paraffin-embedding, a Leica PelorisII tissue processor and Sakura Tissue-Tech embedding station were used. Paraffin section (thickness, 3–5 µm) were produced using ThermoFisher rotary microtome. Cryo- or Paraffin sections were stained with haematoxylin-eosin using an automatic station (Tissue-Tek Prisma), Masson’s Trichrome Kit (Leica, Raymond A Lamb, BDH Chemicals) and Van Gieson Staining Kit (Millipore-Merck).

Atherosclerotic lesions from the heart were cut into 7 mm sections on a Leica 3050 cryostat at −25°C. Cross area sections of 42 mm were stained with toluidine blue (0.2% in PBS, Sigma-Aldrich, Gillingham, UK) to determine lesion size. Total lesion size per section was measured using Adobe Photoshop CS4. Lesion severity was scored (0, 1, 2, 3, 4, 5) by an experienced pathologist as no lesion (score 0) early (intimal xanthoma, scores 1, 2), moderate (pathological intimal thickening, score 3) and advanced (fibrous cap atheroma, scores 4, 5), as described elsewhere (Kanters et al., 2003 (link)). Sirius red staining was performed for 30 min to measure collagen content (0.05% direct red in saturated picric acid, Sigma, Zwijndrecht, Netherlands). Images were obtained using a Leica DM3000 microscope and quantified with Adobe Photoshop CS4 where collagen was quantified as the percentage of total lesion size. For immunohistochemistry, slides were fixed in acetone and blocked with Avidin/Biotin Blocking Kit (Vector Laboratories, Burlingame, USA). Hereafter, cells were incubated with MOMA-2 (1:4000, AbD Serotec, Uden, Netherlands) to stain for macrophages, ER-MP58 (1:200, AbD serotec, Uden, Netherlands) for infiltrating monocytes. Necrosis area was measured based on toluidine blue staining by a pathologist and corrected for total plaque size.

PC3-PIP xenograft tissue from mice was obtained from preclinics GmbH (Germany) and stored at − 80 °C. Tissues were allowed to equilibrate for at least 1 h in the cryotome chamber of a Leica 3050 Cryostat before sectioning at − 18 °C (chamber temperature). Sections with 10 µm thickness were attached to a poly-D-lysine coated petri dish and stored at − 80 °C until use for max. 2 weeks. Prior the assay, the tissue was pre-warmed with the petri-dish being inverted inside an incubator (37 °C, 5% CO₂, saturated humidity) for 5 min. A consecutive slide was cut onto a microscope slide, stained with H&E and imaged with a microscope (Keyence).