Department of Otolaryngology-HNS, Virginia Merrill Bloedel Hearing Research Center, University of Washington, Seattle, Washington 98195-7923, USA.
Microsc Res Tech. 2010 Jan;73(1):37-44. doi: 10.1002/jemt.20750.
The organotypic slice culture (Stoppini et al. A simple method for organotypic cultures of nervous tissue. 1991;37:173-182) 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. Single-cell electroporation for gene transfer in vivo. 2001;29:583-591). The culture was then placed in a chamber perfused with oxygenated ACSF and the labeled cell imaged with an inverted wide-field microscope repeatedly for multiple days, recording several time-points 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 custom-built insulator constructed around the microscope to maintain controlled temperature and uses a perfusion chamber as used for in vitro slice recordings.
器官型切片培养（Stoppini 等人，一种简单的神经组织器官型培养方法，1991；37：173-182）已经成为回答神经科学各种问题的首选方法。然而，对于许多实验来说，如果能够重复地对切片培养进行成像或操作，例如在多天的时间内，将会非常有益。我们制备了 P3 和 P4 小鼠听觉脑干的器官型切片培养物，并在体外培养了长达 4 周。通过电穿孔的方式将表达荧光蛋白的质粒转染到听觉脑干中的单个细胞中（Haas 等人，活体单细胞电穿孔转染，2001；29：583-591）。然后，将培养物置于用含氧 ACSF 灌注的腔室内，并使用倒置宽场显微镜对其进行多次重复成像，每天记录多个时间点，然后再将切片放回孵育器中。我们描述了一种简单的方法，可以在多天和多个连续小时的时间内对切片培养物进行成像，而不会对组织造成明显的损伤或光漂白。我们的方法使用了一个简单、廉价的定制绝缘器，围绕显微镜构建，以维持受控的温度，并使用了用于体外切片记录的灌流室。
