A slit was then made along the dorsal midline using a glass capillary pulled to a sharp point, and the body walls glued flat to the coverslip.
They were subjected to perfusion fixation as follows: after clamping of the ascending aorta and clipping of the right atrium, 1 ml of PEM buffer (1 mM EGTA, 1 mM MgCl2, and 100 mM PIPES, pH 6.9) containing 20 mM KCl was administrated from the left ventricle using a pulled capillary tube, followed by perfusion of 3 ml of 3.7% formaldehyde.
Tapered micropipets with an inner tip diameter of 0.6 ± 0.2 μm were obtained by heat-pulling borosilicate glass capillaries (o.d./i.d. = 1.0 mm/0.58 mm, 10 cm in length, Sutter Instrument, Novato, CA) using a CO2-laser capillary puller (model P-2000, Sutter Instrument).
The patch pipettes (2 4 MΩ resistance when filled with the internal solution) were pulled from Hilgenberg (FRG) or Clark (England) borosilicate glass capillaries using a Zeitz puller (DMZ, Munich, FRG) and filled with the solution I1 for the measurement of APs.
Pipettes were fabricated from borosilicate glass capillaries using a micropipette puller P-20000, Sutter Instrument, Novato, CA, USA) with resistance of 3 5 MΩ when filled with pipette solution.
borosylicate glass capillary tubing using a micropipette puller.
The surrounding fat and tissue were removed using a pulled glass capillary pipette.
MO oligonucleotides were injected into the hindbrain lumen of 22 25 somite-old embryos (14 15 Hamburger Hamilton stage) by using a pulled glass capillary.
The capillary was then pulled using a P-2000 laser puller (Sutter Instrument Co).
Blastocysts (typically 30 50 per experiment) were treated with acid Tyrode's solution to remove the zona pellucida, then incubated in 2 mg/ml collagenase IV for 20 min at 37°C, followed by Hanks'-based cell dissociation buffer (Invitrogen) for 20 min on ice, then manually dissociated into single cells using a finely pulled glass capillary.
