Die Embryonalentwicklung der Paradiesschnecke ... - TOBIAS-lib

Kapitel 2
Scanning electron microscopy
All Petri dishes received eggs from at least three different clutches. Embryos
were fixed at different stages of their development. Every day, beginning with
the first day of the experiment (age 0 day), 10–12 embryos were removed from
their eggs and transferred into fixative (2% glutaraldehyde (VWR-Merck)
dissolved in 0.01 mol l −1 cacodylate buffer (VWR-Merck), pH 7.4). For each
timepoint, two specific Petri dishes were used, one for the control and one
for the platinum exposure. As the eggs were taken from different clutches,
slight differences in age were possible. Used Petri dishes were discarded.
The eggs were opened with two syringes, and the embryos were removed
and then transferred into snap-cap vials using an Eppendorff pipette. The
snap-cap vials were filled with the mentioned fixative. Until further processing
the samples were kept at 4 ◦ C.
Processing started with rinsing the embryos in 0.01 mol l −1 cacodylate
buffer (three times for 10 min each). The embryos were then stained overnight
with reduced osmium tetroxide and dehydrated successively with 75%, 80%,
85%, 95% and absolute ethanol (three times 15 min for each concentration).
The specimens were then critical point dried, mounted on stubs, and sputtered
with gold. They were examined with a scanning electron microscope
(Cambridge Stereoscan 250 Mk2, Cambridge Scientific, Cambridge, UK),
and pictures were taken. The images were edited with Adobe Photoshop
CS2 (Adobe Systems; converting, cropping, and background color), GIMP
2.6 (converting, cropping, and scaling), and Inkscape 0.48 (converting and
labeling) and examined visually.
Histology
Every day, beginning with the first day of the experiment, between six and
fifteen embryos from both control and the platinum group were removed
from the egg capsules (description see earlier) and fixed in Bouin’s solution
overnight or for several days. The embryos fixed on a given day all
derived from the same clutch, assuring that control and platinum embryos
were all of exactly the same age. After fixation, the embryos were washed
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