Abstract
The organotypic slice culture (Stoppini et al. A simple method for organotypic cultures of nervous tissue. 1991;37:173β182) has become the method of choice to answer a variety of questions in neuroscience. For many experiments, however, it would be beneficial to image or manipulate a slice culture repeatedly, for example, over the course of many days. We prepared organotypic slice cultures of the auditory brainstem of P3 and P4 mice and kept them in vitro for up to 4 weeks. Single cells in the auditory brainstem were transfected with plasmids expressing fluorescent proteins by way of electroporation (Haas et al. Singleβcell electroporation for gene transfer in vivo. 2001;29:583β591). The culture was then placed in a chamber perfused with oxygenated ACSF and the labeled cell imaged with an inverted wideβfield microscope repeatedly for multiple days, recording several timeβpoints per day, before returning the slice to the incubator. We describe a simple method to image a slice culture preparation during the course of multiple days and over many continuous hours, without noticeable damage to the tissue or photobleaching. Our method uses a simple, inexpensive customβbuilt insulator constructed around the microscope to maintain controlled temperature and uses a perfusion chamber as used for in vitro slice recordings. Microsc. Res. Tech., 2009. Β© 2009 WileyβLiss, Inc.