Figure 5
Visual confirmation of shRNA delivery into embryos via electroporation. Representative images of 4 hpf embryos are shown for each experimental condition. All images were projected from confocal z-stacks of ~ 20 μm. Hoechst staining of DNA is shown in blue (note some embryonic cells undergoing mitosis) and rhodamine-tyramide in red. The bottom row shows the single red channel in black and white for each condition to enhance contrast. In all cases, shRNAs were delivered at a concentration of 900 ng/μl. (A) Scrambled shRNA-electroporated embryo for which tyramide signal amplification (TSA) was carried out. (B) Digoxigenin-labeled scrambled shRNA-electroporated embryo for which TSA was performed but peroxidase-labeled anti-DIG antibody (Ab) was not added. Note the complete lack of rhodamine-tyramide signal in A,B controls. (C) Embryo that was soaked with digoxigenin-labeled scrambled shRNA for the length of an electroporation procedure (~ 3 min) but was not electroporated, and for which the TSA reaction was carried out. Note that some dots appear with the rhodamine-tyramide signal, mostly from shRNAs stuck on the surface but not inside the cells (see Supplementary Video S3). (D) Digoxigenin-labeled scrambled shRNA-electroporated embryo for which TSA was performed. Notice the intense fluorescence generated from the rhodamine-tyramide signal, largely coming from shRNAs that were delivered inside the one-cell stage embryonic stage via electroporation, and which stayed inside the cells throughout the first stages of cleavage (see Supplementary Video S4). Scale bar 50 μm.
