Die Embryonalentwicklung der Paradiesschnecke ... - TOBIAS-lib

Kapitel 4
in the figures of this publication in blue lines). Additionally, we added nerve
connections that were expected but could not be doubtlessly observed in the
slices directly because some slices were artificially damaged or folded. These
structures were added to the figures in black dashed lines to emphasize that
they are only hypothetical. Image editing was accomplished in Gimp Image
Editor (scaling, cropping, color adjustment) and in Inkscape (labeling).
Immunohistochemistry with M. cornuarietis embryos
After reaching the required age (5 and 6 days for the controls and 7 to 12 days
for PtCl 2 -exposure group), the embryos were removed from the egg capsules
with syringes and transferred into small glass Petri dishes filled with acidulated
water for relaxation for 10 to 15 minutes prior to fixation (Römerquelle
medium, Göppingen, Germany, Ca: 604 mg/L, Mg: 47,3 mg/L, CHO 3− :
203 mg/L, Na: 20 mg/L, Cl − : 6,3 mg/L, SO −4
2 : 1328 mg/L). The embryos
were then fixed in 4% Triton-paraformaldehyde (0.1% Triton X diluted in 4%
paraformaldehyde diluted in phosphate-buffered saline (PBS, 8.0 g/L NaCl,
0.2 g/L KCl, 1.78 g/L Na 2 HPO 4 ∗2H 2 O and 0.27 g/L KH 2 PO 4 diluted in
bidistilled water, pH=7.4)). Fixation lasted at least 12 hours and was conducted
at 6 ◦ C. The embryos were then washed 4 times à 5 minutes in PBZ
(0.1% sodium azide in PBS) and then transferred into a blocking buffer for
4 to 5 hours at 6 ◦ C (blocking buffer: 1% Triton X, 3% horse serum and 10%
of 1% PBZ diluted in PBS). The specimens were then incubated with the
first antibody with a concentration of 1:200 in blocking buffer at 6 ◦ C (rabbit
anti-serotonin whole antiserum (Sigma-Aldrich, St. Louis, MO, USA), polyclonal
rabbit anti-FMRFamide antibody (ImmunoStar, Hudson, WI, USA)
or monoclonal mouse anti-acetylated tubulin antibody (Sigma-Aldrich, St.
Louis, MO, USA)). Preliminary tests showed that incubation times between
48 h and 96 h yielded the same results and we therefore used either 48 h, 72
h, or 96 h for incubation. Subsequently, the embryos were washed 8 times
à 45 min in 0.1% PBZ and then incubated with the second antibody (goat
anti-rabbit IgG antibody linked to the fluorophor Alexa 488 R○ (Invitrogen,
Eugene, OR, USA) with a concentration of 1:200 in blocking buffer for 48,
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