Vacutainer serum blood collection tubes

Venous blood was collected in BD Vacutainer Serum Blood Collection tubes with spray-coated silica as a clot activator and then transported to the University of Oklahoma Integrative Immunology Center (IIC) within two hours of collection. Blood tubes were centrifuged at 1300×g for 10 min at room temperature, serum was removed, aliquoted, and then stored at − 80 °C until analysis.

Prior to euthanasia, animals were weighed. Blood was drawn from the heart, incubated in BD Vacutainer^® serum blood collection tubes for 1 h at room temperarture (RT), and subsequently stored on ice until centrifugation at 1,000× g for 10 min to isolate serum which was immediately delivered to Virginia Tech Animal Laboratory Services (ViTALS) for quantification of cortisol, cholesterol, and glucose levels. Owing to the diurnal nature of cortisol, bood was collected from all animals within each cohort within a 3-h window (9 AM-12 PM).
Quickly following euthanasia, brains were removed from the skull, weighed, and cut at 5 mm intervals on a custom-designed brain mold, and fixed at 4°C in 4% paraformaldehyde (0.1 M PBS) for 72 h. After fixation, tissue slabs were cryoprotected at 4°C in a sucrose gradient of 20% and 30% (0.1 M PBS) until sunken. Tissue was embedded in OCT compound (Tissue-Plus #4585) and stored at −80°C for subsequent serial sectioning on a Thermo Scientific CryoStar NX50 cryostat. Coronal tissue sections (50 μm) were collected for immunohistochemical procedures. For tissue used in H&E and DAB staining, 5 mm tissue blocks were incubated in 10% formalin for 72 h, embedded in paraffin, and cut on a microtome at 5 μm thickness. Histopathological assessments were performed by certified pathologists at Virginia Tech on cadavers at 42 days of age.