Tert amyl alcohol

Animal husbandry was performed according to the guidelines of St. Joseph Hospital and Medical Center and Barrow Neurological Institute under the Institutional Animal Care and Use Committee–approved protocol. Five- to six-week-old IcrTac:ICR-Prkdcscid mice were used for in vivo orthotopic transplant of fluorescently-tagged GB3 cells. For orthotopic transplants, 2 μL of dissociated cells at a density of 50,000 cells μL⁻¹ were injected in the right hemisphere (stereotactic coordinates AP 0, ML −2, DV −2.5), as described previously [42 (link), 43 (link)]. 8 weeks after injection, tumor-bearing mice were euthanized with a lethal intraperitoneal injection of 2.5% Avertin (2,2,2-Tribromoethanol, Sigma-Aldrich; T48402; tert-Amyl alcohol, Sigma-Aldrich, A1685). Tissues were fixed through intracardial perfusion with Ringer’s solution (Electron Microscopy Sciences; 11763–10) supplemented with 40 mM NaNO₂, 2 mM NaCHO₃, and 50 IU mL⁻¹ heparin, followed by ice-cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). Brains were subsequently cryoprotected with incubation in a solution of 30% sucrose in PB for 48 hours before being frozen and cut into 40 μm coronal sections using a cryostat (Microm HM550, Thermo Scientific).

Mice were anesthetized using an intraperitoneal injection of 1.2% Avertin at a dosage of 20 μL per gram body weight. Avertin was prepared by dissolving 2-2-2 tribromoethanol [Sigma, Cat# T48402] in tert-amyl alcohol [Sigma, Cat# 240486] to make a concentrated solution and then dissolved in water to make a 1.25 working solution. Saliva flow was then stimulated using an IP injection of 0.1μg/μL pilocarpine [Sigma, Cat# P6053] at a rate of 5 μL per gram body weight. Saliva flow monitored for ten minutes following pilocarpine injection. Saliva was collected in 20 or 100 μL capillary tubes and measured.