Qf100 70 10

Zygotes were prepared for microinjections with a membrane softening pretreatment. Embryos were exposed to a freshly made 1:1 solution of 1 M sucrose and 0.25 M sodium citrate for 20 s followed by three quick rinses with FSW (Meyer et al., 2010 (link)). Individual zygotes were pressure injected with quartz glass needles (QF100-70-10; Sutter Instruments) that were pulled using a micropipette puller (P-2000; Sutter Instruments). Needles were filled with a cocktail of 800 μM MO antisense oligonucleotides, nuclease-free water and a 1:10 dilution of 20 mg/ml red dextran reconstituted in FSW (Texas Red, Molecular Probes). The volume of microinjected MO cocktail was typically between 0.5 and 2% of the total embryo volume, as estimated from injection into an oil drop, and resulted in a final MO concentration of 4-12 µM. For rescue experiments, needles were filled with 100 ng/μl of Ct-Smad2/3 mRNA plus dextran, 100 ng/μl Ct-Smad2/3 mRNA 3′ 6×His tag plus dextran, or a combination of 100 ng/μl Ct-Smad2/3 mRNA with 800 μM MO plus dextran. Injected and uninjected animals from the same brood were raised in FSW containing 60 μg/ml penicillin and 50 μg/ml streptomycin in separate dishes, and compared to assess overall brood health. A brood was considered healthy if more than 90% of the uninjected animals developed normally.

Quartz glass capillaries used in the experiments (QF100-70-10, Sutter Instrument Co., Novato, CA, USA) have an outer diameter of 1 mm, an inner diameter of 0.7 mm, and a length of 7.5 cm. Before use, the glass capillaries were ultrasonically cleaned in ethanol and pure water for 15 min, and the liquid residue on the tube walls was removed using a nitrogen gas stream. The capillaries were then pulled into nanopores using a CO₂ laser capillary puller (model P-2000, Sutter Instruments Co., Novato, CA, USA) with the parameters shown in Table 1:
The glass nanopore was electrochemically characterized in electrolyte (1 M KCl, 10 mM Tris-HCl, pH = 8.0) using cyclic voltammetry. The ion current was measured at intervals of 100 mV in the range of −500 mV to +500 mV, and an I-V scan curve was plotted accordingly. The conductance value of the nanopore was determined using the slope of the curve, and the diameter of the glass nanopore was calculated. Subsequently, the morphological characteristics and diameter of the glass nanopore were characterized using scanning electron microscopy (SEM) to verify the accuracy of the nanopore diameter estimation obtained using electrochemical methods.

Conical glass nanopores were made from quartz glass capillaries (O.D: 1 mm; I.D: 0.7 mm; QF100-70-10; Sutter Instrument Co.). All glass capillaries used in the experiments were thoroughly cleaned by immersing them in freshly prepared piranha solution (3 : 1 98% H₂SO₄/30% H₂O₂) for ∼2 h to remove organic impurities. (Caution: piranha solution is a powerful oxidizing agent and reacts violently with organic compounds. It should be handled with extreme care). The capillaries were rinsed thoroughly with deionized water and vacuum dried at 70 °C prior to use. Glass nanopores were then fabricated using a CO₂-laser-actuated pipette puller (model: P-2000, Sutter Instrument Co.) with an on-line program involving the following parameters: heat = 760; fil = 4; vel = 31; del = 120; pul = 170. TEM images were obtained using an FEI TECNAI F20 EM with an accelerating voltage of 200 kV. The tip (∼2 mm) of a nanopipette was cut off and transferred to a copper grid for TEM imaging.