The overall goal of this procedure is to assess the spatial temporal expression of specific genes of interest within staged zebrafish embryos. This is accomplished by first generating CD NA probes by incorporating digo oxygen in linked nucleotides using in vitro transcription. Next, the embryos are harvested at defined developmental stages.
The corium are removed and the embryos are stored in methanol at negative 20 degrees Celsius. Then following digestion and post fixation, the embryos are hybridized with the appropriate probes. Finally, the embryos are stained with antide gene and antibodies and the signal developed with substrates for alkaline phosphatase.
Ultimately, results can be obtained that show the tissue distribution of gene expression at a given developmental stage through whole mount in situ hybridization and imaging. Though this method can provide great greater insights into the normal expression of target gene, it can also be applied to other conditions such as target gene expression in disease model, and when other genes are over or under expressed. Using microinjection technology Using CDNA for a gene of interest, set up an RNA labeling reaction to synthesize digo oxygen In labeled RNA probes in an RNAs free reaction vial by adding one to five micrograms of purified template DNA to sterile RNAs free dey treated double distilled water up to a volume of 13 microliters.
Chill the reaction on ice and add the following reagents after gently mixing centrifuge 2000 RPM for 30 seconds at room temperature before incubating the reaction for two hours at 37 degrees Celsius after the incubation, remove the template DNA by adding two microliters of RNAs free. DNAs one incubate for 30 minutes at 37 degrees Celsius and stop the reaction by adding two microliters of 0.2 molar EDTA, precipitate the reaction products by adding 2.5 microliters of four molar lithium chloride and 75 microliters of 100%ethanol. Mix the solution and incubate at negative 20 degrees Celsius for 45 minutes or overnight.
Centrifuge the suspension for 30 minutes at 13, 000 RPM and four degrees Celsius. Then wash the pellet with ice cold, 70%ethanol after spinning for 10 minutes. Dry the pellet for two minutes before resus, suspending it in 50 microliters of nuclease, free double distilled water.
Then incubate it for 30 minutes. At 37 degrees Celsius, run one to two microliters of the probe on a 1%aros gel for about 30 minutes to verify its quality and integrity. Then use a spectrophotometer to quantify the probe to rehydrate previously fixed and frozen embryos.
Begin by making baskets by cutting the conical ends off of 1.5 milliliter micro tubes. Then remove the top rims from half of them. Cut small pieces of nylon mesh to cover the ends, and then in a few hood, heat the tube ends on an electrical hot plate and press them into the mesh after they've cooled.
Remove the excess nylon mesh and store the baskets in 100%methanol at room temperature. After preparing successive dilution of methanol in PBS, according to the text protocol and adding four milliliters of each into the wells of a six well dish place embryos in individual baskets for each probe and incubate them in the methanol PBS series for five minutes per well ending with three washes in PBST. Perme the embryos with proteinase K at room temperature, using the following incubation times per developmental stage.
Then fix the embryos in 4%paraform aldehyde in PBS for 20 minutes and wash them in PBST three times for five minutes each. Next, prepare the acetylation mixture according to the text protocol and incubate the embryos twice for 10 minutes each. Then wash the embryos and PBST twice for 10 minutes each.
Transfer the embryos to sterile micro tubes and incubate with 200 microliters of hybridization mixture or HM, in a 70 degree Celsius water bath for two to four hours. Add 100 to 200 nanograms of probe to the solution and incubate overnight at 70 degrees Celsius in a water bath. Replace the probe solution with pre warmed HM and incubate the samples at 70 degrees Celsius for 15 minutes.
After putting the samples through an H-M-S-S-C series and blocking the embryos according to the text protocol, incubate the embryos in a one to 5, 000 dilution of anti-D AP antibody in blocking buffer overnight at four degrees Celsius with gentle agitation the following day. Wash the embryos six times for 15 minutes each. After pres staining the embryos, incubate them in BCIP and NBT.
Add room temperature in the dark for 30 minutes. Use a stereo microscope to monitor the staining reaction every 30 minutes to stop the reaction. Transfer the baskets of embryos to wells containing stop solution two times for 10 minutes each with gentle agitation.
After post fixing the embryos with 4%para formaldehyde in PBS for 20 minutes. Mount the embryos by transferring them in as little PBS as possible to six well plates containing 100%glycerol. Gently rock the embryos in the dark at room temperature, overnight image in 100%glycerol under a stereo microscope.
Representative examples of situ to hybridization staining are shown here. Purple staining observed after hybridization with ribo probe of interest represents both the abundance and the sites of expression of a particular gene. For instance, PC 5.1 shows very discrete expression within the anterior and posterior part of otic vesicle and lateral line primordial at 24 hours post fertilization and is strongly localized within the anterior and posterior lateral line neuro masts by 72 hours post fertilization.
In contrast, PC 5.2 shows you ubiquitous expression within the CNS with distinct regionalization within the somites otic vesicle and prone fric duct, and is highly expressed within the liver and intestine at 96 hours post fertilization. Absence of gene expression when using respective sense Ribo probes serves as a negative control as seen here. Expression analyses of blood markers showed staining of SCL T one within the bilateral cranial cells and in the intermediate cell mass or ICM.
Although the staining for gata one is also seen within the ICM, the expression pattern is different. The expression of F-L-K-K-D-R-L was observed within the hind brain main and intersegmental vessels, and in the ICM staining for sonic hedgehog was observed in the no cord. In these examples expression of DLX two at 34 hours post fertilization is localized in the Talon Cephalon hypothalamus and cranio mesenchymal arches and the FK D seven Fox, a one expression at 24 hours post fertilization is seen mainly in the floor plate and HypoChlor expression.
Patterns of the pancreatic markers showed insulin staining in the endocrine pancreas and trypsin in the exocrine pancreas. The results showing expression patterns with high resolution confirm the success of the outlined protocol for the detection of the expression of both high and low abundance genes shown. Here are some examples from experiments carried out under some optimal conditions.
For example, a high background signal with poor sonic hedgehog expression resulted from inadequate, permeable and hybridization between 55 and 60 degrees Celsius. After watching this video, you should have a good understanding of how to perform whole mount in two hybridization of zebra fish embryos using ditch labeled RNA probes, post hybridization staining and imaging.
