Fig. 1. Microinjection and electroporation system for kidney organ cultures. (A) A diagram of the microinjection and electroporation system. (a) A three-dimensional depiction, showing a pair of electrodes on the left, and the injection needle on the right. (b) A view from above the organ culture; showing the injection needle placed in the condensed mesenchyme. The dotted line shows the boundary of the whole organ culture. Injections can also be placed outside the condensed mesenchyme, within the looser mesenchyme of the organ culture. (c) Whole-mount double exposure of brightfield image of organ culture and GFP fluorescence 24 hours after injection. (B-E) Gdnf injection/electroporation. (B,D,E) A Gdnf expression vector was injected outside the condensed mesenchyme, adjacent to the Wolffian duct, as shown by the arrow in B. (C) An empty vector was injected as a negative control. (B,C) Cytokeratin stains done 36 hours post-injection to demonstrate the presence of ectopic ureteric buds emerging from the Wolffian duct. Several are present in B, none in C, next to the yellow arrows. (D,E) In situ hybridization for Gdnf, using antisense (D) and sense (E) probes. The endogenous Gdnf mRNA signal is too weak to be detected under these hybridization conditions. CM, condensed mesenchyme; UB, ureteric bud.