4.1 Materials
4.2.4 Molecular analysis of gene expression
4.2.4.1 Whole-mount in situ hybridization
This method was used to detect the spatio-temporal expression patterns of genes involved in neural crest specification and differentiation. Zebrafish embryos are transparent until the melanophores start to produce melanin around 26 hpf. We incubated developing embryos in 0.2 mM PTU (1-Phenyl-2-thio-urea) starting at 8-10 hpf in order to prevent melanin synthesis and thus maintain optical clarity of the embryos.
Staged embryos were fixed in 4% PFA (paraformaldehyde) overnight at 4°C. They were dechorionated and rinsed in PBT (PBS with 0.1% Tween-20) several times. The duration of the following permeabilization step with proteinase K varied depending on the age of the embryos:
<tail bud no digestion
0-2 somites 10 µg/ml prot. K, 2 min. 4-10 somites 10 µg/ml prot. K, 3.5 min. 10-20 somites 10 µg/ml prot. K, 5 min. 24 hpf 10 µg/ml prot. K, 10 min. 48 hpf 10 µg/ml prot. K., 30 min. > 48 hpf 50 µg/ml prot. K, 1 hour
Following 2 quick washes with PBT, the embryos were refixed with 4% PFA for at least 20 minutes. After rinsing 5 times in PBT, the embryos were prehybridized for at least one hour in hybridization mix at 70°C in a waterbath.
Hybridization mix: 65% formamide 32.5 ml 100% stock (hybe mix) 5x SSC 12.5 ml 20x SSC stock
50 µg/ml heparin 86 µl of 29 mg/ml 0.5 mg/ml yeast tRNA 25 mg powder
0.1% Tween-20 250 µl 20% stock
9.2 mM citric acid, pH 6.0 920 µl 0.5 M stock 3.8 ml H2O
50 ml total
For hybridization, the embryos were incubated in 250 µl of new hybridization mix containing 500 ng of digoxygenin-labeled riboprobe. The hybridization step was
Materials and Methods
performed overnight at 70°C in a waterbath. The following day the embryos were rinsed as follows:
10 min. 70°C in 75% hybe mix + 25% 2x SSC 10 min. 70°C in 50% hybe mix + 50% 2x SSC 10 min. 70°C in 25% hybe mix + 75% 2x SSC 10 min. 70°C in 100% 2x SSC
30 min. 70°C in 0.05x SSC 30 min. 70°C in 0.05x SSC
5 min. room temperature in 75% 0.05x SSC + 25% PBT 5 min. room temperature in 50% 0.05x SSC + 50% PBT 5 min. room temperature in 25% 0.05x SSC + 75% PBT 5 min. room temperature in 100% PBT
Afterwards the embryos were incubated in the blocking solution PBT/NGS/BSA for at least one hour at room temperature. PBT/NGS/BSA is PBT containing 2% NGS (normal goat serum) and 2 mg/ml BSA (bovine serum albumin).
The anti dig-AP (alkaline phosphatase) antibody (Boehringer Mannheim) was pre- adsorbed before usage. For this purpose it was diluted 1/100 in PBT/NGS/BSA and incubated overnight at 4°C in a tube containing zebrafish embryos. The antibody binding step with the pre-adsorbed anti dig-AP antibody (diluted 1:50 in PBT/NGS/BSA) was performed overnight at 4°C.
Embryos were rinsed at room temperature 5 times in PBT for 5 min each and 3 times in NTMT for 15 min each.
NTMT: 100 mM Tris-HCl pH 9.5 5 ml 1M stock
50 mM MgCl2 2.5 ml 1M stock
100 mM NaCl 1 ml 5M stock
0.1% Tween-20 250 µl 20% stock
+ dH2O to 50 ml total volume
The alkaline phosphatase staining was developed at room temperature in freshly prepared NTMT/NBT/BCIP solution in the dark.
NTMT/NBT/BCIP: 10 ml NTMT
225 µg/ml NBT 22.5 µl (100 mg/ml in 70% DMF) 175 µg/ml BCIP 35 µl (50 mg/ml in 100% DMF)
Materials and Methods
The reaction was monitored under the stereo microscope until adequate staining was visible (routinely 30 min to 2 hours, depending on individual probes and the stage of the embryos). The embryos were rinsed four times in PBT, fixed in 4% PFA, rinsed again 2 times in PBT and then rinsed in PBT + 5 mM EDTA for 20 min. After the washes, the embryos were transferred to 80% glycerol/20% PBT to clear and they were stored at 4°C.
4.2.4.2 Whole-mount immunohistochemistry
To monitor protein expression in whole mount embryos, I performed immunohistochemistry using the mouse monoclonal antibody anti-Hu 16A11 (anti- human neuronal protein HuC/HuD) from Molecular Probes.
Solutions: PTD: PBS 0.3% Triton X-100 1% DMSO PTDBN: PTD
2 mg/ml BSA (bovine serum albumin) 2% NGS (normal goat serum)
The zebrafish embryos were fixed overnight in 4% PFA, rinsed in PBT and then dehydrated in a methanol/PBTseries:
15 min 25% MeOH / 75% PBT 15 min 50% MeOH / 50% PBT 15 min 75% MeOH / 25% PBT 15 min 100% MeOH
The embryos were stored overnight at –20°C in 100% methanol. This step contributes to the permeabilization of the embryos.
Materials and Methods
The rehydration series has been done in reverse order to the above dehydration series:
15 min 75% MeOH / 25% PBT 15 min 50% MeOH / 50% PBT 15 min 25% MeOH / 75% PBT 15 min 100% PBT
Subsequently, the embryos were bleached with 1 ml 10% H2O2 + 1 drop of 2 M KOH
in an open tube for 40 min. Following three 5 min washes with PBT, the embryos were permeabilized by incubation in 0.1 mg/ml proteinase K solution for 10 min at room temperature. After two washes with PBT for 1 min each, the embryos were refixed with 4% PFA for 20 min. Following five 5 min washes in PBT, 2 quick washes in distilled water and 2 rinsing steps in acetone, the embryos were frozen in acetone at -20°C for 1 hour. This step enhances the permeabilization effects achieved by the methanol and proteinase K treatments. Then two quick washing steps with distilled water and five washing steps with PTD for 5 min each were performed. After this the embryos were transferred to 24 well plates and incubated in the blocking solution PTDBN (freshly prepared) for at least 1 hour. The primary antibody, mouse anti-Hu, was diluted (1:20) in PTDBN. The embryos were incubated in this solution overnight at 4°C.
The following day, after five washes with PTD for 10 min each, the secondary antibody (anti-mouse IgG diluted 1:200 in PTDBN) was applied to the embryos. Following a 1 hour incubation at room temperature, five washes in PTD for 10 min each had to performed. In the meantime the ABC complex (Vectastain kit) was prepared: This was done by mixing 20 µl solution A (avidin) with 1 ml PTDBN, then adding 20 µl solution B (biotinylated-HRP) and mixing again. The ABC complex was prepared at least 30 min before usage. The incubation in the ABC complex was for 1 hour at room temperature. Five 10 min washes in PTD preceded the staining procedure, which was performed as follows using the Vectastain kit:
Two drops (100 µl) of buffer stock solution were mixed with 5 ml distilled water. Four drops (200 µl) of DAB (diaminobenzidine) stock solution were added, followed by mixing. The embryos were preincubated in one half of the above solution for 10 min in the dark. One drop (50 µl) of H2O2 solution was mixed with the remaining solution.
Materials and Methods
staining solution. The developing reaction was allowed to occur in the dark till adequate staining was achieved (routinely 1 - 40 min). Subsequently the staining reaction was stopped by rinsing the embryos in distilled water for at least 20 min. The embryos were transferred to PBT and finally to the mounting medium 80% glycerol/20% PBT. The embryos were stored in this medium at 4°C.
Materials and Methods