Beuthansia

At PND 25 – 26, blood was collected from the submandibular vein via puncture with a lancet at a depth of ~1 mm. At the end of study (PND 42), mice were euthanized by intraperitoneal (IP) injection of Beuthansia at 150 mg/kg (Merck & Co., Kenilworth, NJ, USA) as described previously (45 (link), 47 (link), 48 (link)). Thereafter, blood was collected from the right atrium and was refrigerated at 4°C for an hour. Blood was then centrifuged at 2,000 × rpm to collect sera and was then stored at –20°C for further analysis. Following blood collection, mice were perfused with a 1 × PBS solution through the left ventricle. Then, thoracic spinal cord and hippocampus were removed and flash-frozen in liquid nitrogen. These tissues were selected for analysis based on their known involvement in TMEV infection (49 (link)–51 (link)). Tissues were stored at -80°C for RNA extractions, as described below.

Mice were anesthetized with Beuthansia (0.2 ml, i.p.; Merck) and perfused transcardially with PBS followed by 4% PFA in PBS. Brains were post-fixed overnight in 4% PFA at 4 °C, cryoprotected in 30% sucrose, frozen in OCT compound (ThermoFisher), and stored at −80 °C. Coronal sections (30 μm) were cut on a cryostat (Leica Microsystems) and collected in cold PBS. For immunohistochemistry experiments, sections were washed three times in PBS with 0.2% Triton X-100 (PBST) for 5 min and incubated in blocking solution (3% normal donkey serum in PBST) for 1 h at room temperature. Sections were incubated overnight at 4 °C in blocking solution with primary antibodies including chicken-anti-GFP (1:10000, Abcam, ab13970) and rabbit-anti-dsRed (1:2000, Takara Bio, 632496). After 3 washes in PBS, sections were incubated for 1 h in PBS with secondary antibodies: Alexa Fluor 488 donkey anti-chicken, Cy5 donkey anti-chicken, Alexa Fluor 594 donkey anti-rabbit, and/or Cy5 donkey anti-rabbit (1:500, Jackson ImmunoResearch). The tissue was washed 3 times in PBS, mounted onto glass slides, and coverslipped with Fluoromount-G (Southern Biotech). Fluorescent images were acquired using a confocal microscope. All digital images were processed in the same way between experimental conditions to avoid artificial manipulation between different datasets.