Glass capillary

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.

GABA-mediated inhibitory postsynaptic currents in miniature (mIPSCs) were recorded using the patch-clamp technique in the whole cell voltage-clamp configuration in the presence of 1 µM tetrodotoxin (TTX, Tocris) to block the generation of action potentials. Recordings were obtained from primary hippocampal neurons at DIV14 (plated on poly-L-lysine coated coverslips at the density of 234 cells/mm²) using the Axopatch 200B amplifier and the pClamp-10 software (Axon Instruments). Recordings were performed in Krebs’-Ringer’s-HEPES (KRH) external solution (mM: 125 NaCl, 5 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 2 CaCl₂, 6 glucose, 25 HEPES-NaOH pH 7.4). Recording pipettes were pulled from glass capillary (World Precision Instrument) using a two-stage puller (Narishige) and had tip resistances of 3–5 MΩ when filled with the intracellular solution (mM: 130 Cs-gluconate, 8 CsCl, 2 NaCl, 10 HEPES, 4 EGTA, 4 MgATP, 0.3 GTP pH 7.3). Voltage-clamp recordings were performed at holding potentials of +10 mV. The recorded traces were analysed using Clapfit-pClamp 10 software, after choosing an appropriate threshold. In particular, events that exceeded at least twice the standard deviation of the baseline noise were considered as mIPSCs and included in our analyses.

Membrane potential was measured with the ruptured whole-cell patch clamp. Microelectrodes were pulled from glass capillary (World Precision Instruments, Sarasota, FL, USA) by a pipette puller (Sutter Instrument, Novato, CA, USA) and polished with a micro forge (NARISHIGE, Japan). The microelectrodes used in experiments were typically 3–6 MΩ after they were filled with the internal solution. Both microelectrodes and solutions were prepared freshly before experiments. Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) was used to amplify the signal detected by the microelectrode. pClamp 10.4 software (Molecular Devices) was used for recording signals. Action potential (AP) data were analyzed with Cardiac Action Potential Analysis Software (CAPA) Package Distributed by Science Consulting Cardiac Cellular Electrophysiology UG (Essen, Germany) [23 ].

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.

Adult females with ages ranging from 5 to 22 days old were blood-fed using a glass water jacketed membrane feeder. The next day, females were immobilized by incubation at 4° until motionless, then placed on ice and sorted. Females with visible blood-meals were injected intrathoracically using a glass needle drawn from a glass capillary (World Precision Instruments) using a needle puller (Sutter P2000) and aspirator assembly (A5177, Sigma) until we could observe visible distention of the abdomen, diuresis or liquid emerging from the injection site (approximately 200 nl).