Fluorescent Whole-Mount Method for Visualizing Three-Dimensional Relationships in Intact and Regenerating Adult Newt Spinal Cords

Adult newts have the remarkable ability to regenerate their spinal cords after a complete transection injury. To understand this process, we have developed a method for visualizing the cellular and molecular events during regeneration in whole-mount preparations using fluorescent probes (streptavidins and antibodies) and confocal microscopy. This method was optimized by varying parameters associated with fixation, tissue trimming, fluorescent probe penetration, and clearing and represents a significant advance in our ability to observe the intact and regenerating newt spinal cord. These methods should also be widely applicable to the study of other newt tissues and adult tissues from other model systems. Developmental Dynamics 239:3048-3057, 2010. (C) 2010 Wiley-Liss, Inc.