Peri star pro

At 7 days post-TETS exposure, a subset of mice imaged in this study was anesthetized with 4–5% isoflurane in medical-grade oxygen, perfused with ~25 mL of 4% (w/v) paraformaldehyde (PFA; Sigma; St. Louis, MO, USA) in phosphate-buffered saline (PBS; 3.6 mM Na₂HPO₄, 1.4 mM NaH₂PO₄, 150 mM NaCl; pH 7.2) using a Peri-Star Pro peristaltic pump (5 mL/min). Brains were removed, and the freshly perfused whole brains were immediately placed into 20 mL glass scintillation vials containing 10 mL of 4% (w/v) PFA in PBS (pH 7.2). After 24 h, whole brains were transferred into 30% (w/v) sucrose (Fisher; Houston, TX, USA) in PBS (3.6 mM Na₂HPO₄, 1.4 mM NaH₂PO₄, 150 mM NaCl; pH 7.2) for an additional 48 h or until the tissue sank completely to the bottom of the vial. Fixed whole brains were serially sectioned into 2-mm thick coronal slices using a mouse brain matrix (Zivic Instruments, #5325; Pittsburgh, PA, USA) and embedded by flash freezing in optimal cutting temperature medium (OCT; Fisher HealthCare; Waltham, MA, USA). Blocked brain sections were stored at −80 °C until cryosectioned using a Microm HM550 cryostat (Thermo Fisher Scientific, Waltham, MA, USA) into 10-μm thick slices on Superfrost plus microscope slides (Fisher HealthCare). Slides were stored at −80 °C prior to staining or immunohistochemistry.