PhD thesis

Material and methods
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Detection of proliferating cells with BrdU (5-bromo-2-deoxyuridine)
staining
BrdU labeling was carried out, in order to identify possible growth zones in
rhynchonelliform brachiopod larvae. BrdU is incorporated into the DNA of
proliferating cells during the S-phase of the cell cycle. Staining of BrdU thus
allows for visualization of dividing cells and their progenies. Larvae of Terebratalia
transversa of the following developmental stages: 6, 11, 24, 35, 48, 60, and 96
hours after fertilization (hpf) were incubated in 0.1mM BrdU (Sigma-Aldrich,
St. Louis, MO, USA) in seawater at 11.5ºC for 6 – 48h. In another experiment
larvae were cultured in 10mM BrdU in seawater for 30 min and subsequently
the larvae were cultured in BrdU free seawater (pulse-chase experiment). After
the treatment with BrdU the larvae were fixed in 4% paraformaldehyde in PBS
for 1 hour at room temperature and then treated for 10 min at 37ºC in 0.01mg/
ml proteinase K in PBS. After that they were kept for 10 min in 0.1N HCl on
ice, 1 hour at 37ºC in 2N HCl, 1 hour in PBS with three changes, and 15 min in
PBT (PBS with Tween 20). Then, the larvae were incubated in 1:500 mouseanti-BrdU
antibody in PBT over night at 4 ºC, washed for 1 hour in PBS with
three changes, 1 hour in 1:200 diluted TRITC, and finally 1 hour in PBS with
three changes. Stained larvae were mounted in glycerol and analyzed with a
Leica DM RXE 6 TL fluorescence microscope equipped with a TCS SP2 AOBS
laserscanning device (Leica Microsystems, Wetzlar, Germany).
Gene expression analyses
The expression of developmental genes was studied by whole mount in
situ hybridization (WMISH). Thereby, target mRNA is visualized with a
complementary RNA probe which contains DIG labelled uridine (Digoxigenin-
11-uridine-5’-triphosphate). The digoxigenin is subsequently stained with a
Anti-DIG-AP, fab fragments antibody that contains alkaline phosphatase (AP)
which in turn is made visible by a reaction with BCIP (5-Bromo-4-chloro-3-
indolyl phosphate) and NBT (nitro blue tetrazolium chloride). In this reaction
BCIP is dephosphorylated by AP and dimerizes to leucoindigo. This dimer is
then oxidized by NBT to an insoluable dark blue 5,5’-dibromo-4,4’ precipitate
(Trinh et al. 2007). The precipitate is visible in daylight conditions and also
reflects laser light which allows the use of this technique in combination with a
confocal laserscanning microscope (Jekely and Arendt 2007).
There are several WMISH protocols available which usually have to be adapted
to the organism they are intended for. Protocols developed for several species
were tested in this study, namely one for the sea urchin Strongylocentrotus