Microinjection holding pipet

Fig. 7. Labeling of the epiblast by microinjecting Dil into the distal cap of the embryo. To inject in the midline, the embryo is held at the distal tip of the egg cylinder hy gentle suction with the holding pipet (h). The injection pipet (ip) is passed through the extraembryonic tissues of the egg cylinder. The injection pipet is brought to the site of labeling from within the pro-amniotic cavity to avoid inadvertent labeling of other embryonic germ layers. The arrow points to the tip of the labeling pipet in the epiblast layer en, primitive endoderm primitive streak. Bar = 20 pm.

Micromanipulators Two are required—for the holding pipet, which holds the egg in place, and for the microinjection pipet. The excellent Leica mechanical micromanipulator (Wetzlor, Germany), with joystick control of the horizontal movement in two planes, is most commonly used. The micromanipulators and the microscope must be positioned on a purpose-built base plate (Fig. 5), which must be custom engineered. Narishige (Tokyo, Japan) produces economically priced micromanipulation systems compatible with Nikon microscopes. These are very flexible and do not require a custom-built base plate. 

Fig. 5. A typical arrangement of the equipment needed for the microinjection of fertilized one-cell eggs. A Agla micrometer S5ringe. B Liquid-paraffin-filled tube. C Left-hand micromanipulator. D Inverted microscope. E Base plate. F Camera (optional). G Left-hand instrument tube for holding pipet. H Microinjection chamber (depression slide) sitting on fixed stage. I Right-hand instrument tube for injection pipet. J Air-filled tube. K Glass 50-mL syringe. L Right-hand micromanipulator. M Video system (optional).

Move the capillary to a horizontal position. Position the filament 1-2 mm below the capillary tip. Move the filament close to the pipet, and heat to bend the capillary by about 15° to the horizontal. One holding pipet lasts one microinjection session and is not reused. Large numbers can be made in advance and stored in a sterile plastic tube. 

Position the microinjection pipet until the tip is 2cm above the center of the microinjection chamber and 15-20° to the horizontal. Using the fine vertical control of the micromanipulator, lower the tip into the injection chamber until it is just above the chamber floor. Monitor the position of the tip throughout using the 4x objective. Using the horizontal controls of the nuCTomanipulators, bring both the injection pipet and the holding pipet to the center of the field of view. 

Switch to the 40x Nonnarski objective, and focus on the holding pipeL Using the line controls of the micromanipulator, bring the tip of the microinjection pipet into the same focal plane as the holding pipet. The microinjection pipet should move freely in the horizontal plane when operated by the joystick control. 

Switch back to the 4x objective, and place the injected egg above the holding and microinjection pipet. Injected eggs should be divided into two groups eggs that have survived injection and those that have not. 

---