Die Embryonalentwicklung der Paradiesschnecke ... - TOBIAS-lib

Kapitel 1
for 4 × 5 min with PTw (PBS with 0.01% Tween 20, Roth, Karlsruhe, Germany)
embryos were incubated with PTw+N (PTw with 5% Goat Serum,
Jackson ImmunoResearch, West Grove, PA, USA) overnight at 4 ◦ C.
For horseradish peroxidase (HRP) reaction embryos were washed with
PTw for 4 x 5 min and 4 x 15 min and afterwards incubated in 0.3 mg/mL
DAB solution for 20 min. The reaction was started by adding H 2 O 2 to
a final concentration of 0.03%. When the brown signal became visible, as
could be visually detected under a stereomicroscope, reaction was stopped
by washing 2 × 5 min with PTw. Finally, embryos were mounted in a
50% glycerol/DAPI (4’,6-diamidino-2-phenylindole) mix. For visual analysis
and photography were used a light microscope and a stereomicroscope (both
Zeiss, Jena, Germany).
Synchrotron X-ray phase contrast tomography and
holotomography
Whole intact embryos of M. cornuarietis exposed to 100 µg/L PtCl 2 for 26
dpf were fixed in 100% ethanol overnight, critical point dried (CPD 020,
Balzers, Wiesbaden, Germany) and mounted on specimen holder stubs. X-
ray tomography was conducted at beamline ID19 (ESRF, Grenoble, France)
at an energy of 20.0 keV. Measurements were performed at three different
sample-detector distances, i.e. 16 mm, 100 mm, and 841 mm. The effective
pixel resolution amounted to 5.05 µm. Otherwise, instrument settings and
further data treatments were done according to the description of Heethoff
and Cloetens (2008). Due to limitations in beam time, measurements were
conducted with a single animal each of the Pt treated group and of the
control group.
Scanning electron microscopy
Embryos of M. cornuarietis of different age and exposure (100 or 200 µg/L
PtCl 2 , and controls) were fixed overnight in 2% glutaraldehyde in 0.01 M
cacodylate buffer at pH 7.4. Subsequently, the organisms were washed three
times with 0.01 M cacodylate buffer and stored in 1% osmium tetroxide
39