Perfusion setup for imaging adult rodent brain tissue slices on an inverted microscope platform. Warm perfusate is maintained in a covered reservoir (1-L glass bottle) sitting within a warm water bath. For perfusates requiring oxygenation, a tube (white arrowhead) is run from the oxygen tank (95% O2, not shown) into the reservoir perfusate, and the perfusate is continuously aerated with the gas. Perfusate is delivered to and removed from the specimen chamber using a two-channel variable-speed pump (black arrowhead; e.g., Bioptechs) that maintains a constant rate of perfusate delivery and removal (∼1 mL/min). The specimen chamber (white arrow) holds ∼2 mL of perfusate. The tubes running to and from the specimen chamber are taped to the mobile portion of the microscope stage to minimize drag. Used perfusate is aspirated from the chamber and pumped into a waste container. Care must be taken to avoid dripping or spilling perfusate on any microscope or electronic components. Here, a custom-made Plexiglas shield (beneath the pump) was built to prevent perfusate from leaking onto electronic components in the confocal scan head.
