P 2000 pipette puller

Single barrel: pH-sensitive nanomembrane probes were fabricated by pulling a borosilicate glass capillary (O.D. 1 mm, I.D. 0.58 mm) to micropipettes with a tip diameter of ~1 µm or nanopipettes with a diameter of ~100 nm using a laser-based puller (Model P-2000, Sutter Instruments Co., USA). Nanopipettes were pulled with a two-step protocol. Briefly, for the initial step, the parameters were: heat 350, filament 3, velocity 30 and delay 200. For the second step, they were: heat 350, filament 2, velocity 27, delay 160 and pull 250. It should be noted that these values are instrument-specific, and parameters would have to be optimised for each instrument in order to obtain similar nanopore.
Double barrel: Double-barrel pH nanoprobes were constructed from a double-barrel quartz theta capillary (O.D., 1.2 mm, I.D., 0.9 mm, Sutter Instruments), which was pulled with a laser-based P-2000 pipette puller (Sutter Instruments) using a single line program (heat 700, filament 3, velocity 45, delay 130, and pull 93) to produce sharp double-barrel nanopipettes. The size of each barrel of this pulled double-barrel nanopipette was about 100 nm.

To perform gene transfer experiments in vivo, P1 pups were anesthetized and injected via the posterior semicircular canal (PSCC) technique as previously described.⁶² (link) Briefly, injection was performed using beveled glass microinjection pipettes, which were pulled from capillary glass on a P-2000 pipette puller (Sutter Instruments). Pups were anesthetized by rapid induction of hypothermia for 3–4 min on ice until loss of consciousness, and this state was maintained on a cooling platform for 10–15 min during the surgery. The surgical site was disinfected by scrubbing with Betadine and wiping with 70% ethanol. A postauricular incision was made to expose the PSCC and penetrate the tip of the micropipette. A total volume of 1 μL of either virus was unilaterally introduced at a rate of 300 nL/min into the left ear. The skin incision was closed using superglue. Body temperature was maintained on a 37°C warming pad for 30 min after surgery and before reintroduction into the parental cage.

Mouse pups were injected via the round window membrane (RWM) technique using beveled glass microinjection pipettes on postnatal day (P) between P0 and P30 as indicated. Pipettes were pulled from capillary glass on a P-2000 pipette puller (Sutter Instruments). Pups were anesthetized by rapid induction of hypothermia for 2–4 min on ice water until loss of consciousness, and this state was maintained on a cooling platform for 10–15 min during the surgery. The surgical site was disinfected by scrubbing with Betadine and wiping with 70% Ethanol. A post-auricular incision was made to expose the transparent otic bulla, and the RWM was penetrated by the tip of the micropipette. Approximately 1 μL of virus was slowly introduced unilaterally into the left ear. The skin incision was closed using a 6-0 monofilament suture (Ethicon). EMLA cream (lidocaine 2.5% and prilocaine 2.5%) was applied externally for analgesia using sterile swabs to cover the surgical site (left mastoid prominence). Body temperature was maintained on a 37 °C warming pad for 30–60 min after surgery and before reintroduction into parental cage.