Pc 10 puller

Microinjection of pre-blastoderm embryos was performed essentially as described in [70 (link)] with little modifications. Females of the Oregon-R strain were allowed to lay eggs for one hour on grape juice agar plates. Eggs were washed with a 70% ethanol (v/v) solution, and aligned manually on a coverslip, mounted on a microscope slide, briefly desiccated, covered with halocarbon oil and injected at either their posterior or anterior pole with a capillary needle attached to an Eppendorf Femtojet microinjector. Needles for microinjection were obtained from borosilicate glass capillaries, pulled with a Narishige PC-10 puller. Concentration of injected DNA was usually 0.5 to 0.8 mg/ml. After injection, the cover slip containing the embryos were carefully removed from the slides and transferred to grape juice plates. After incubation at 18°C for 24 hours, embryos were further subjected to RNA extraction.

Cells in the retina were bulk electroporated as described (Ruthazer et al., 2006 (link)). Cells were imaged roughly 48 hr after electroporation. In brief, glass micropipettes were made from borosilicate capillaries pulled on a PC-10 puller (Narishige, East Meadow, NY). Pipette were loaded with plasmid solution and attached to a custom-made pressure injection system. Plasmid solution (0.5–5 µg/µL) was then pressure-injected in the eye, without visibly distending the eye, and current was delivered across custom-made platinum plate electrodes placed on either side of the eye using 3 pulses (36 Volts, 1.6 ms) in each polarity using a constant voltage stimulator (Grass SD-9) with a 3 µF capacitor placed in parallel.

Whole-cell voltage-clamp experiments were performed following the procedures described previously (Hamill et al., 1981 (link)), using an EPC-9 amplifier (HEKA Eletronik, Germany) at room temperature (21–25°C). Patch pipettes were fabricated from glass capillary tubes by PC-10 Puller (Narishige, Japan) with the resistance of 2–3 MΩ. Data acquisition and stimulation protocols were controlled by a Pentium III computer (Legend, Beijing, China) equipped with Pulse/PulseFit 8.3 software (HEKA Eletronik, Germany). Capacitance transients were cancelled. Cells with a seal resistance (Rseal) below 1 GΩ were omitted. Series resistance (Rs) was compensated (80–85%) to minimize voltage errors, and cells with an uncompensated Rs above 10 MΩ were omitted. Leak subtraction was performed using P/6 protocol. Data were low-passed at 10 kHz. Unless stated specially, for HEK 293T cells, the holding potential was −80 mV. BK channel currents were elicited by the step pulses ranging from −100 to +150 mV for 200 ms with the increments of 10 mV. The holding potentials were held at −80 mV for BK channel. Current density calculation formula (pA/pF), where pA represents the current of BK channel and pF represents the membrane area of measured cell.

Recording electrodes were pulled from borosilicate glass capillaries (10–15 MOhm, BioMedical Instruments) with a PC-10 puller (Narishige) and then filled with 2 M NaCl solution, in which 2% HRP (Type II, Sigma-Aldrich) was dissolved. First, the electrode was stereotactically aligned above the IC coordinates (Figure 1), and then progressively lowered down in the tissue while presenting white noise to the animal. A careful approach to the recorded neuron guaranteed measurements of spikes from single neurons. In most parts of the IC, a crisp background response of Multi-Unit-Activity (MUA) to the noise stimulus could be monitored; recordings were considered to be single unit activity, when action potential amplitude was at least three-fold the signal-to-noise ratio (snr) of the local MUA. At the end of each recording session the neighboring neurons were labeled with an injection of HRP by three 90 V DC current injections over 3 min (ISO-STIM 01D, NPI electronic).

Whole-cell current clamp recordings were made in pyramidal cells of the OFC. Pyramidal cells that expressed hM4Di were identified by the fluorescent mCherry label using an Olympus BX51WI microscope mounted on a vibration isolation table and a high-power LED (LED4D067, Thor Labs). Recordings were made in ACSF containing (in mM): 120 NaCl, 25 NaHCO₃, 1.23 NaH₂PO₄, 3.3 KCl, 0.9 MgCl₂, 2.0 CaCl₂, and 10 dextrose, bubbled with 95% O₂/5% CO₂. ACSF was continuously perfused at a rate of 2.0 mL/min and maintained at a temperature of 32°C. Picrotoxin (50 µM) was included in the recording ACSF to block GABA_A receptor-mediated synaptic currents. Recording electrodes (thin-wall glass, WPI Instruments) were made using a PC-10 puller (Narishige International, Amityville, NY) to yield resistances between 3–6 MΩ. Electrodes were filled with (in mM): 135 KMeSO₄, 12 NaCl, 0.5 EGTA, 10 HEPES, 2 Mg-ATP, 0.3 Tris-GTP, 260–270 mOsm (pH 7.3). Access resistance was monitored throughout the experiments. Cells in which access resistance varied more than 20% were not included in the analysis.

To evaluate the biodistribution of nanoemulsions in vivo, 48 hpf zebrafish embryos were microinjected in the duct of Cuvier with TopFluor-labelled PSN (with and without Cy5-labelled miR/pDNA) previously concentrated 10 times by the SpeedVac Concentrator (Savant SPD111V-120, Cambridge Scientific, Massachusetts, United States). The microinjection was carried out with a binocular loupe (SMZ745, Nikon), the IM 300 Microinjector (Narishige, Tokyo, Japan), and needles made with the PC-10 Puller (Narishige, Tokyo, Japan) from glass capillaries (Harvard Apparatus, Massachusetts, United States). After 48 h from the microinjection, embryos were processed as explained in Section 2.9 and nanoemulsions biodistribution was evaluated by confocal microscopy.