Article Figures & Data
Figures
- Figure 1.
A, Schematic drawing of the cross section of the capillary gun. B, Photograph of the gun with a head attached, mounted on a micromanipulator, and aimed at a leech embryo. Inset, Close-up view of the leech embryo with a laser spot (red dot); the end of the gun is seen extending from the top right corner. C, Distributions of penetration depths of 1.6 μm gold particles in leech embryos. The particles were shot at He pressures and distances between the nozzle and the embryo of 14.5 psi and 2 mm, respectively (black curve), and of 10 psi and 4 mm, respectively, (gray curve). The distributions were obtained by fixing the tissue and counting particles at different depths under a dark-field microscope, as described in detail by Rinberg et al. (2005).
- Figure 2.
Bright-field micrographs of portions of fixed and processed leech embryos. The embryos in A–C and E–G were stained by in situ hybridization with a netrin probe. A, Three midbody segments of an untreated E10 embryo, showing the normal distribution of netrin mRNA (dark bands) in ventral longitudinal muscle cells (some indicated by arrows) and in neurons in two central ganglia (enclosed by dashed lines). B, An E10 embryo shot with netrin dsRNA-coated particles using the gun. The shot areas are enclosed by white dashed lines. Note the absence of hybridization signals in a number of muscle cells within the encircled areas. C, Close-up image of the area of a shot. Arrows point at cells with netrin mRNA knockdown. D, An embryo stained by hybridization labeling for Act3 constitutively expressed by leech muscle cells. Regular staining in the shot area (white dashed circle) indicates normal expression of actin mRNA in the cells bombarded by netrin dsRNA-coated particles. E, An untreated ganglion with netrin mRNA expressed in several neurons (dark spots). F, A ganglion after one shot with netrin dsRNA-coated particles, showing visible reduction in netrin mRNA. G, A ganglion that received three shots of the particles, showing complete silencing of netrin expression. The small dark dots in F and G are gold particles. The ganglia in E–G are from the same embryo. Scale bars, 100 μm.
- Figure 3.
Transfection of central neurons and a longitudinal muscle cell by particles coated with a plasmid encoding EGFP-Act1 and injection of two different lipophilic dyes. (For the details of microscopy, see supplemental material, available at www.jneurosci.org). A, Superimposed stack of fluorescence confocal sections of a dorsal region of an embryonic ganglion, 48 h after a shot. Cell nuclei were counterstained with a nonspecific nucleophilic compound (Hoechst, blue). Expression of EGFP-Act1 is clearly seen in the cell bodies and neurites of three neurons (there were two other expressers in different focal planes). Scale bar, 10 μm. Inset, Superimposed stack of reflected confocal sections taken in the same planes of the same ganglion; arrows indicate particles. B, Longitudinal muscle cell expressing EGFP-Act1, imaged with a confocal microscope 48 h after a shot. Inset, Magnified image of a fragment of the same cell taken with a transmission filter; one can see a particle in the cell nucleus (red arrow). Scale bar, 10 μm. C, Fluorescence image showing superposed stacks of confocal sections taken at conditions optimized for detection of green and red fluorescence in a dorsal region of an E10 embryo containing two segmental ganglia. The imaging was performed 2 h after particles coated with green (DiO) and red (DiI) fluorescent dyes were shot into the upper and lower ganglia, respectively. One can see some diffusion of dyes within cells. Scale bar, 100 μm. Inset, The same type of confocal image taken in a ganglion of an E10 embryo immediately after particles coated with the red and green dyes were shot into it; the gun was displaced by ∼100 μm between the shots.
Additional Files
Supplemental data
Files in this Data Supplement:
- supplemental material - Supplemental material
- supplemental material - FIGURE LEGENDS Supplemental Figure 1: Schematic drawing showing the pneumatic capillary gun with a head attached to it mounted on a micromanipulator. Arrows indicate the flow of He.
- supplemental material - Supplemental Figure 2: Transfection and staining of mammalian cells cultured in a Petri dish using particles coated with a plasmid encoding GFP and with a lipophilic dye. A and B, Fluorescence image and a superposition of brightfield and fluorescence images of a cell culture ~24 hr after receiving a shot of particles coated with plasmid DNA encoding GFP. The black dots in panel B are gold particles. The cell layer is nearly confluent and is not visibly damaged by the shot. Expression of GFP is clearly seen in 5 cells. The images are taken with a 10� objective, scale bar 100 μm. C and D, Magnified images of a portion of the area shown in panels A and B, with two transfected cells. Fluorescence image in C and a superposition of brightfield and fluorescence images in D. In addition to the cell bodies, expression of GFP is clearly seen in processes of the cells. Images are taken with a 50� objective, scale bar 10 μm. E and F, Fluorescence image and a superposition of brightfield and fluorescence images of a cell culture after a shot of particles coated with a red fluorescent lipophilic dye (DiI). The number of gold particles (black dots) is much smaller than in panels A-B, and just as in panels A-B, the cell layer is nearly confluent and undamaged. The images are taken with a 10� objective, scale bar 100 μm. G and H, Magnified images of a portion of the area shown in panels E and F , fluorescence in G and a superposition of brightfield and fluorescence in H, with three fluorescently stained cells. All fluorescent cells have gold particles inside. The images are taken with a 50� objective, scale bar 10 μm.
