Biology Reference
In-Depth Information
over the low-melt agarose to spread it out before it solidifies. Spacers can be
used on either side of the slide to obtain the correct thickness. Use the edge of
a slide resting on two spacer slides on either side of the slide containing
embryos to apply pressure to the coverslip and compress the agarose to a
uniform thickness. Ideal mount thickness is 100 m m, which is the thickness
of the smallest HPF specimen carrier configuration. To obtain agarose of the
right consistency, heat the low melting temperature agarose to boiling in a
glass test tube. The agarose is most easily dispensed by cutting 5-10 cm off the
end of a yellow pipette tip and preheating the tip by rotating in the hot agarose.
Fill the tip with 70 m L of hot agarose and then dispense the agarose in a line
along the edge side of the slide, over the high-strength agarose. The actual
volume of agarose dispensed on the pad is less than 70 m L, as about half (or
more) of the agarose stays in the tip. Use the 70 m L as a starting point and
adjust as needed. This small volume cools rapidly, so the coverslip must be
quickly placed over the low melting temperature agarose and gently pressed
down to spread a thin layer of agarose around the embryos. A microscope slide
turned on edge can be used to apply pressure to the coverslip on either side of
the embryos but not directly over the embryos. The top layer of agarose is
thinner than the high melting temperature agarose used to make the base. If the
mount is too thick, embryos will be beyond the focal depth of the confocal
microscope, making it impossible to acquire a fluorescent image. Note that
covering embryos with hot low melting temperature agarose does not work
with embryos that have been bleached, because they are too fragile.
iv. Seal the edges of the slide with Valap to prevent dehydration.
v. Acquire a focal series through the group of embryos at 1 m m intervals using
the appropriate excitation wavelength (488 nm for GFP). A transmitted light
image can be acquired simultaneously with each fluorescent image of GFP
expression. In our experiments, embryos must attain a minimum age to be
able to identify ''mutant'' from rescued embryos. The transmitted light image
can be used to confirm the developmental stage of embryos.
b. HPF and Freeze Substitution
i. Configure HPF specimen carriers to provide a 100- or 200- m m deep well.
To allow access to the agarose-immobilized embryos, scrape the Valap off
the slide (which has already been imaged in a confocal microscope), and
push the coverslip horizontally off the agarose pad using a single-edged
razor blade. Cut out a small square of the agarose including the embedded
embryos (maximum size is 2 mm diameter) and transfer this small pad to a
bottom specimen carrier using a razor blade. The agarose pad can be
pushed into a specimen carrier using a sharpened toothpick that has been
coated with 1-hexadecene to keep the toothpick from adhering to the
agarose pad. Loading tiny samples into specimen carriers becomes a battle
with surface tension. Adhering carriers to the tops of Petri dishes with
double stick tape holds carriers in place during the loading process. Double
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