7. Murray’s clear (also known as BB:BA): 2 volumes of benzyl
benzoate and 1 volume of benzyl alcohol: optional for clearing
a tissue.
2.3 Tools
1. Hair loop and hair knife (looped hair or eye brow hair fixed to
the end of pulled Pasteur glass pipette with melted wax).
2. Disposable plastic pipette—Cut the opening of a disposable
pipette to enlarge the opening hole to freely transfer embryos,
aggregates, or embryonic cells with minimal shear stresses.
3. Forceps (Dumont #5 stainless steel; Fine Science Tools or
equivalent).
4. Cover glass (various sizes; #1 1/2 thickness).
5. Silicon grease (high vacuum, Dow Chemical or equivalent).
6. PCR tubes. To prevent cell adhesions to the wall of PCR tube,
add 200 μl of 1% BSA to PCR tube and coat for overnight at
4
C.
2.4 Microinjection
1. Borosilicate glass capillary tubes (1.0 mm OD Â 100 mm).
2. Needle puller (p-97, Flaming/Brown Micropipette Puller, Sutter Instrument Co or equivalent).
3. Microinjector (PLI-100, Harvard Apparatus or equivalent).
4. Micromanipulator (M33, Marzhauser or equivalent).
5. Microloader Pipette Tip.
6. mRNA transcription kit (Epicenter or equivalent).
7. Mineral oil.
8. Horizontal graduated scale eyepiece reticle.
2.5 Imaging
1. Imaging chambers (see Note 1 and Fig. 1).
2. Electron microscope grid (optional): Useful for positioning
arrays of aggregates within imaging chambers.
3. Stereoscope/Fluorescent stereoscope: Essential for microinjection and screening injected embryos and aggregates.
4. Confocal microscope. Laser scanning or spinning disk
mounted on inverted compound microscope. High numerical
aperture objectives such as 63Â oil, or 25Â water immersion
are useful to track protein dynamics. Longer working distance
objectives are helpful for live imaging and collecting image
stacks for 3D reconstruction. A motorized z-stage is essential
while a motorized xy-stage is optional.
5. Image analysis software. Essential for post processing image
stacks and quantifying epithelial areas and cell dynamics.
FIJI/ImageJ or equivalent.
278
Hye Young Kim and Lance A. Davidson
benzoate and 1 volume of benzyl alcohol: optional for clearing
a tissue.
2.3 Tools
1. Hair loop and hair knife (looped hair or eye brow hair fixed to
the end of pulled Pasteur glass pipette with melted wax).
2. Disposable plastic pipette—Cut the opening of a disposable
pipette to enlarge the opening hole to freely transfer embryos,
aggregates, or embryonic cells with minimal shear stresses.
3. Forceps (Dumont #5 stainless steel; Fine Science Tools or
equivalent).
4. Cover glass (various sizes; #1 1/2 thickness).
5. Silicon grease (high vacuum, Dow Chemical or equivalent).
6. PCR tubes. To prevent cell adhesions to the wall of PCR tube,
add 200 μl of 1% BSA to PCR tube and coat for overnight at
4
C.
2.4 Microinjection
1. Borosilicate glass capillary tubes (1.0 mm OD Â 100 mm).
2. Needle puller (p-97, Flaming/Brown Micropipette Puller, Sutter Instrument Co or equivalent).
3. Microinjector (PLI-100, Harvard Apparatus or equivalent).
4. Micromanipulator (M33, Marzhauser or equivalent).
5. Microloader Pipette Tip.
6. mRNA transcription kit (Epicenter or equivalent).
7. Mineral oil.
8. Horizontal graduated scale eyepiece reticle.
2.5 Imaging
1. Imaging chambers (see Note 1 and Fig. 1).
2. Electron microscope grid (optional): Useful for positioning
arrays of aggregates within imaging chambers.
3. Stereoscope/Fluorescent stereoscope: Essential for microinjection and screening injected embryos and aggregates.
4. Confocal microscope. Laser scanning or spinning disk
mounted on inverted compound microscope. High numerical
aperture objectives such as 63Â oil, or 25Â water immersion
are useful to track protein dynamics. Longer working distance
objectives are helpful for live imaging and collecting image
stacks for 3D reconstruction. A motorized z-stage is essential
while a motorized xy-stage is optional.
5. Image analysis software. Essential for post processing image
stacks and quantifying epithelial areas and cell dynamics.
FIJI/ImageJ or equivalent.
278
Hye Young Kim and Lance A. Davidson