PhD thesis
PhD thesis
PhD thesis
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66 Submitted manuscript<br />
Chapter IV<br />
Netherlands) and subsequently ligated<br />
into a pGEM-T Easy vector (Promega,<br />
Madison, WI, USA). Ligation products<br />
were transformed into One Shot<br />
TOP10 E. coli competent cells<br />
(Invitrogen, Carlsbad, CA, USA). Cells<br />
were allowed to grow over night; clone<br />
DNA was isolated using a QIAprep<br />
Spin Miniprep Kit (Qiagen). Insert<br />
sequences were sequenced at the<br />
sequencing facility of the University<br />
of Barcelona and identified using<br />
the tBLASTx algorithm. Two specific<br />
forward primers were subsequently<br />
designed from the TtrNot and the<br />
TtrCdx PCR sequences: TtrNotF1 5’-<br />
GGA GAA GGA GTT CGA AAG GCA<br />
ACA A-3’, TtrNotF2 5’-CCG AAT CCC<br />
AAG TGA AGA TCT GGT-3’, TtrCdxF1<br />
5’-CCT GGA GCT GGA GAA GGA<br />
GTT CTG T-3’, and TtrCdxF2 5’-AAC<br />
AAC CTT GTA CTT TCA GAG AGA<br />
CAG G-3’. The specific primers were<br />
used nested in a 3’RACE-PCR using<br />
a SMART RACE kit following the<br />
manufacturer’s protocol (Clontech,<br />
Mountain View, CA, USA). The<br />
sequences of the RACE-PCR products<br />
were again checked by BLAST and the<br />
positive clones were used for in situ<br />
probe production using the DIG RNA<br />
Labeling Kit (SP6/T7, Roche, Basel,<br />
Switzerland).<br />
In situs were done following a standard<br />
protocol with a 5 min proteinase K step<br />
and at least 48 hours of hybridization<br />
time at 40ºC or 45ºC (Martindale<br />
et al. 2004; Hejnol and Martindale<br />
2008). For cohorts aged 0-64 hours<br />
after fertilization (hpf), in situs were<br />
performed on developmental stages<br />
that were 2-4 hours apart, for the age<br />
group of 64-154 hpf, in situs were<br />
done every 5-10 hours, while for later<br />
stages longer intervals were chosen.<br />
The latest stages investigated were<br />
540 hpf old, which corresponded to<br />
420 hours after settlement/onset of<br />
metamorphosis (hps). Sense probes<br />
were generated as controls for in situ<br />
hybridization. Since they didn’t give<br />
any signal they are omitted in the<br />
figures.<br />
Stained specimens were photographed<br />
with a Leica ProgRes C3 digital<br />
camera mounted on a Leica MZ<br />
16F stereomicroscope. Schematic<br />
illustrations were generated using<br />
Adobe Illustrator CS3 and CS4<br />
graphics software (Adobe, San Jose,<br />
CA, USA). Analysis of gene sequences<br />
and primer design was done with CLC<br />
Main Workbench 5 (CLC bio, Aarhus,<br />
Denmark).<br />
Immunostaining and confocal<br />
laserscanning microscopy<br />
Larvae were stained with antibodies<br />
against serotonin (ImmunoStar,<br />
Hudson, WI, USA) and acetylated<br />
α-tubulin (Sigma-Aldrich, St. Louis,<br />
MO, USA). In addition, cell nuclei<br />
were labeled using DAPI (Invitrogen,<br />
Eugene, OR, USA). Prior to staining,<br />
larvae were washed thrice for 15min<br />
each in phosphate buffer (PB) and<br />
incubated for 1h in PB containing<br />
0.2% Triton X-100 (Sigma-Aldrich)<br />
at room temperature. Thereafter,<br />
the larvae were incubated over night<br />
at 4ºC in 6% normal goat serum<br />
in 0.1M PB and 0.2% Triton X-100<br />
(blocking solution). Then, the larvae<br />
were incubated for 24 hours at 4ºC in<br />
blocking solution containing a 1:800<br />
dilution of the polyclonal serotonin<br />
antibody, 3µg/ml DAPI, and a 1:800<br />
dilution of the monoclonal acetylated