Physiologic materials:
Zebrafish: Tubingen species is used for the research in the laboratory as an animal model. This one is used because its gene is totally sequenced, It’s also easy to grow in special pools in the laboratory, with high production rate (at least 200 egg per week). In addition this animal is transparent and has an external fecundation which make it easier to manipulate and observe its development. With a high rate of gene conserved between it and humans, it makes it an ideal model to do this king of study.
The system of growing those fish is well developed. It insure the best conditions for the fish. Temperature (28° C), conductivity (200µS), PH (between 7 and 8) and the duration of day-night is (14h-10h) all along the year.
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etc), and so reveal the volume and the development stage of it.
Probes preparation:
RNAm had been synthesized in vitro before I start my stage. The principal thing is that the RNAm contain a nucleotide that marked with the Digoxygenine (DIG), which can be detected by a specific antibody.
Permeabilization and hybridation of Embryos (Day1):
Embryos that were stored in methanol will be recuperate and checked for quantity and quality under a microscope.
Later they will be rehydrated by being placed in baskets and into a 24-well plats. In each well of this plat we will load solutions with decreased methanol concentration (75%, 50%, 25%) diluted in 1 volume of PBS for 5min in each. Then we wash our embryos in PBT (PBS+ Tween) solution 4 times. Tween will help as a siege between the proteins of the membrane.
Permeabilization: embryos will be digested by the Proteinase K for different period of incubation depending on the development stage, (1 cell stage: 30S, 18 somite stage: 10min). We stop the reaction by incubating the embryos for 20min in 4%PFA in PBS, and then washing by the
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Embryos will be incubated in the hybridization mix (deionized formamide, SSC, Tween, Heparin, tRNA, and PH adjustment at 6.0 by Citric acid) for 2 to 5 hours at 70°C.
Embryos at this stage could be stored in HM at -20°C for up to several weeks.
Hybridization:
Discard the last HM and replace it with a new one added to 30-50 ng of antisense or sense DIG-labelled RNA probe.
Hybridization will be done over-night at 70°C (the appropriate temperature for RNA hybridization) in the hybridization oven.
Washes and incubation with anti-DIG antibody alkaline phosphatase (Day2):
After a whole night incubation, HM solution will be removed and embryos will be washed with a decreased HM concentration solutions as followed (75%, 50%, 25% HM), SSC100% twice. These solutions have to be at 70°C before using. Then embryos will be transformed into small baskets and wash them again with decreased solution of SSC and PBT (75%, 50%, 25%), then 100% PBT, which will help cleaning and keeping the pores open.
After then embryos will be incubated in blocking buffer (Sheep serum + BSA) for 3-4 hours at room temperature, on a horizontal