Minipuls 3 peristaltic pump

Cells were sorted using a 100-μm nozzle, on a FACSAria III (BD Biosciences) equipped with four lasers. Fluo-4 AM, Alexa Fluor 594, DAPI, Alexa Fluor 647, and APC-Cy7 were excited at 488 nm, 561 nm, 405 nm, and 633 nm, respectively, and detected using BP530/30, BP610/20, BP450/40, BP660/20, and BP780/60 filters. The sorter was implemented with a homemade injection system (Fig. 1A). The sample line was improved, upstream of the solenoid valve, with an injection system composed of two syringes controlled by valves and a peristaltic micropump. D-PBS was placed in the first syringe. A 1.6× concentrated agonist solution (100 mM KCl, 200 μM AMPA, or 10 μM ionomycin) was prepared, and APC-CY7–labeled CompBead compensation particles (BD Biosciences) were added to the solution before putting it in the second injection syringe. Both the MINIPULS 3 peristaltic pump (Gilson) and the cytometer flow rate were set to 39 µL/min. Baseline acquisition and sorting were performed with the valve of the buffer syringe opened. Once the valve of the agonist syringe was opened, the valve of the buffer syringe was closed. The agonist solution running in the flow cell was monitored by the appearance of the beads. At this point, the agonist-responding cells started to be sorted. Cells were collected in D-PBS. Data were analyzed using BD FACSDiva v6 and FlowJo softwares (BD Biosciences).

To increase the signal/noise ratio and reduce the effective longitudinal relaxation time (T1) in the MRI, mice were perfused using the active staining technique (34 (link)), in which a contrast agent is used in conjunction with perfusion fixation. Mice were sedated by intraperitoneal injection of ketamine (Ketaminol vet solution from Intervet, 50 mg/kg) and xylazine (Rompun vet solution from Bayer Animal Health, 50 mg/kg). Systemic perfusion was performed via the right jugular vein and the left carotid artery with a perfusion pump (MINIPULS 3 Peristaltic pump; Gilson Inc.) at 0.5 mL/min (24 rpm). Vasculature was first flushed with 25 mL phosphate-buffered saline (PBS) at room temperature and thereafter perfused with 20 mL of 4% Histofix (Histofix 4%; Histolab) containing 10% gadoteridol (Prohance solution for injection 279.3 mg/mL, 50 mM; Bracco Sweden). After perfusion, heads were removed and cleaned from skin, eyes, mandible, muscles, and teeth. Next, heads were immersed in 4% Histofix containing 1% gadoteridol for 24 h at 4 °C. Heads were transferred to 0.1 M PBS containing 1% gadoteridol at 4 °C for 7 d. The final step of rehydration in PBS minimized shrinkage or swelling.