Eg1160

Eight-week-old male mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg body weight) and perfused from the left ventricle with 0.1 M phosphate-buffered saline (PBS), pH 7.4 for 10 min and then with 4% paraformaldehyde in PBS for 10 min. Thymic tissues collected from the mice were fixed with 4% paraformaldehyde in PBS for 24 h at room temperature (RT) and then embedded in paraffin using a tissue processor (TP1020; Leica Microsystems, Wetzlar, Germany) under the following conditions: 70% (v/v) ethanol; 80% ethanol; 90% ethanol; three washes with 100% ethanol; three washes with xylene; and embedding twice in paraffin wax for 1 h each time. The FFPE blocks prepared using a paraffin-embedding device (EG1160; Leica Microsystems) were sectioned on a sliding microtome (SM2000R; Leica Microsystems) equipped with disposable blades (S35; Feather Safety Razor, Osaka, Japan). For LMD, 5-μm-thick tissue sections were mounted on poly-l-lysine-coated PEN- and PPS-membrane glass slides (Leica Microsystems). For immuno- and lectin histochemistry, 2-μm-thick sections were placed onto Matsunami Adhesive Slide (MAS)-coated glass slides (Matsunami, Osaka, Japan). The sections were dried overnight at 42 °C and stored at RT until use.

Tasmanian devil tissues were fixed in 10% Neutral Buffered Formaldehyde (Australian Biostain, Traralgon, VIC, Australia) for 24 hr and selected tissues were cassetted (Techno Plas, St. Marys, SA, Australia) and processed overnight using a standard 15-hr overnight procedure in the TP 1050 tissue processor (Leica Microsystems, Wetzlar, Germany). Tissues were orientated on the EG1160 (Leica Microsystems), embedded in paraffin wax (Leica Microsystems) and sectioned at 3 microns using a Leica RM2245 microtome and adhered to microscope slides (Menzel Gläser, Thermo Fisher Scientific) for 20 min at 60°C. Sections were deparaffinized, rehydrated and stained using Jung autostainer XL (Leica Microsystems) for Hematoxylin (Australian Biostain) and Eosin, dehydrated, cleared, cover slipped (Leica Microsystems) and mounted in CV Mount (Leica Microsystems) (Hayes et al., 2017 (link)).