Vertical pipette puller

To observe DNA synthesis by oncogenic Ras isoforms, the microinjection of recombinant Ras proteins has been described previously [21 (link)]. Briefly, CV-1 cells were grown on 12 mm glass coverslips for 24 hr and serum-starved for 24 hr. Cells were microinjected by using glass capillary needles made with a vertical pipette puller (David Kopf Instruments). 2 hr after microinjection, injected cells were treated with BrdU and further incubated for 16 hr at 37°C. After fixed with 3.7% formaldehyde, cells were subsequently incubated with rat anti-BrdU antibody, TRITC-conjugated anti-rat antibody, and FITC-conjugated anti-rabbit IgG antibody. In injection experiments, results represent the mean of at least three independent experiments in which at least 100 cells were injected. To observe cellular localization, CV-1 cells were grown on 12-mm glass coverslips and cotransfected with HA-H-Ras^G12V and GFP-K-Ras^G12V expression vector or HA-H-Ras^G12V and GFP-N-Ras^G12N expression vector for 24 hr. After incubation with serum-free DMEM for 20 hr, cells were fixed, subjected to immunostaining with anti-HA antibody, and imaged with confocal microscope (Carl Zeiss).

A glass capillary of 3 inches length, OD 1.0 mm, with no filament (World Precision Instruments, Sarasota, Florida, USA) was pulled by a vertical pipette puller (David Kopf Instruments, Tujunga, California, USA). The tip was then clipped using a sharp straight Noyes Scissors 4.7 (World Precision Instruments, Sarasota, FL), and loaded with 5 μl of the VMO/ASO-hybrid prepared as described above. Three days post fertilization (dpf) larvae were anesthetized by transferring them using a plastic transfer pipette into a 0.64 mM Tricaine (pH 7.0) and incubating it for 5 seconds. The anesthetized larvae were laid on a 1.2% agarose plate and injected intravenously into the common cardinal vein with 15 nl of VMO/ASO-hybrid using Picospritzer III (Parker Precision Fluidics, Hollis, NH) and a micromanipulator under a dissection microscope. Following the injection, the larvae were transferred to E3 embryonic medium in a plastic cup at 28°C and incubated for 48 hours for use in laser-induced arterial thrombosis assay.

A glass capillary of 3 inches length, OD 1.0 mm, with no filament (World Precision Instruments, Sarasota, Florida, USA) was pulled by a vertical pipette puller (David Kopf Instruments, Tujunga, California, USA). The tip was then clipped using a sharp straight Noyes Scissors 4.7 (World Precision Instruments, Sarasota, FL), and loaded with 5 µl of the VMO/ASO-hybrid prepared as described above. Three days post fertilization (dpf) larvae were anesthetized by transferring them using a plastic transfer pipette into a 0.64 mM Tricaine (pH 7.0) and incubating it for 5 seconds. The anesthetized larvae were laid on a 1.2% agarose plate and injected intravenously into the common cardinal vein with 15 nl of VMO/ASO-hybrid using Picospritzer III (Parker Precision Fluidics, Hollis, NH) and a micromanipulator under a dissection microscope. Following the injection, the larvae were transferred to E3 embryonic medium in a plastic cup at 28 °C and incubated for 48 hours for use in laser-induced arterial thrombosis assay.