This procedure allows us to determine patterns of gene expression in zebrafish embryos at high resolution, an antisense ribo probe is hybridized to the complementary target mRNA. The labeled Ribo probe is recognized by an antibody that is conjugated to a peroxidase enzyme aramide conjugated fluoro four is added to the embryos and the peroxidase converts the IDE to a highly reactive compound. Leading to stable deposition of the fluoro four unbound IDE is washed away revealing the detailed subcellular pattern of mRNA distribution.
Expression of a second gene can be observed using a distinctly labeled probe and a different tyrom fluoro four, allowing the detailed comparison of expression. Hi, I'm Tim Brent from the laboratory of Scott Holly in the Department of Molecular, cellular and Developmental Biology at Yale University. Today we will show you a procedure for whole mount high resolution double fluorescent in situ two hybridization of zebra zebrafish embryos in all laboratory.
We use this procedure to study zebrafish segmentation. So let's get started. Begin the procedure with embryos that were fixed overnight in the cold in para formaldehyde.
Using a freshly thawed stock of paraform aldehyde for this fixation is critical for successful staining. Remove the fixing solution and wash the embryos at room temperature twice for five minutes each time in PBS. Next, remove the corion of the embryos.
The embryos are placed in glass depression plates. Using watchmaker forceps, pull off the Corian from now on. Use a fire polished glass pasture pipette to transfer the embryos.
Otherwise, the embryos will stick to the regular polypropylene tips. The dechlorinated embryos are transferred through a series of washes of increasing methanol concentration. The last wash is in 100%methanol.
Lastly, the embryos are placed in fresh methanol and stored at minus 20 degrees overnight. The next day, transfer the embryos through a series of washes of decreasing methanol concentration and with two five minute PBST buffer washes. Next, fix the embryos and para formaldehyde for 20 minutes.
End the short fixation with two five minute PBST washes. We are now ready to perme the embryos. The fixed embryos are permeable by incubating them with protein ACE K.Make sure to lay the micro centrifuge tube on its side.
The length of the digestion step depends on the age of the embryo. For example, somato agenesis stage embryos are incubated for three to four minutes. Rinse briefly in PBST and wash once in PBST for five minutes.
Fix the embryos in para formaldehyde. Finally wash the embryos twice in PBST for five minutes. The embryos are now ready for hybridization with the RNA probes.
Now that we've fixed and permeable the embryos, we begin the hybridization process with a five minute high minus incubation at 65 degrees Celsius. Remember to preheat the HIIN solution before use. Next, incubate the embryos in high plus for one hour.
Again, remember to preheat the high plus solution. Now remove most of the hip plus solution, but leave 50 microliters, making sure the embryos are completely submerged. Add one microliter of each probe and mix by gently flicking the tube.
Cover the tubes with aluminum foil from now on. Since the fluorescein is light sensitive and incubate overnight at 65 degrees Celsius, the hybridization is done. So let's remove the excess probes from now on.
Detergent is eliminated in order to improve the staining. However, notice that the embryos become sticky in the absence of detergent. Wash twice with 50%form amide two XSSC solution for 30 minutes each time.
Wash once and two XSSC solution for 15 minutes. Finally, wash twice with 0.2 times SSC for 30 minutes each time. Next, we will visualize each probe.
Now we can begin detection of the hybridized probes. Detect the fluorescein probe first because it is less stable and weaker than the dig oxygen and probe. First block with 500 microliters of 2%blocking reagent in maleic acid buffer for one hour at room temperature.
Next, add the peroxidase conjugated anti fluorescein antibody at a one to 500 dilution in the blocking solution. Incubate overnight at four degrees Celsius. Make sure to lay the micro centrifuge tube on its side.
Next, start the detection using the tyrom signal amplification system or TSA TYROM IDE is conjugated to a fluoro four. The horse radish peroxidase on the antibody converts the TY IDE to a highly reactive intermediate. The reactive TY IDE forms a covalent bond to residues in close proximity to the antibody resulting in localized deposition of the fluoro four.
Start with four 20 minute washes with one x maleic acid buffer at room temperature washed twice in PBS. The fluorescein TY IDE was previously dissolved in DMSO for storage. Give this TSA substrate a quick spin down.
Next, dilute the TSA substrate one to 50 in the amplification diluent buffer. Add the solution to the embryos and incubate for 45 minutes. Lay the micro centrifuge tube on its side.
During this incubation step, transfer the embryos through a series of 10 minute washes of increasing methanol concentration. The last wash is 10 minutes in 100%Methanol incubate for 30 minutes in a solution of 1%hydrogen peroxide to inactivate the peroxidase on the antibody. Otherwise, it may interfere with the detection of the dig oxygen in.
Probe transfer the embryos through a series of washes of decreasing methanol concentration and with two 10 minute PBS buffer washes. This ensures that all of the methanol is removed. Antibody labeling of the dig xigen and probe starts with blocking as with the previous antibody.
Next, add the anti dig oxy genin antibody at a one to 1000 dilution. Lay the micro centrifuge tube on its side and incubate overnight in the cold the next day. Wash your room temperature with maleic acid buffer.
Then with PBS quickpin, the SCI five tyrom substrate. Dilute it one to 50 and add it to the embryos. Incubate for 45 minutes laying the tube on its side.
Finally, wash three times in PBST. A final staining step with prop iodide marks the nuclei. Start with two five minute washes in two XSSC.
Next, treat with RNAs for 30 minutes at 37 degrees. Wash six times in two XSSC at room temperature and now add the propidium iodide solution and stain for eight minutes. Wash six times in two XSSC.
Fix the embryos in para formaldehyde for 20 minutes. And finally, two five minute washes in PBST. Mounting the embryos for viewing starts with two 10 minute incubations in 25%and 50%glycerol.
Next, put the samples in 75%glycerol and incubate them overnight at four degrees Celsius. This overnight clearing step makes dissection of the embryos easier. Finally, dissect and remove the yolk from the embryos.
In order to reduce background fluorescence flat mount the embryos on a microscope slide and examine them under the microscope. These images show a single confocal section through the posterior tip of a zebrafish embryo at the 10 So mite stage. The red staining shows delta C mRNA detected with a dig oxygen and labeled probe and TSA sci-fi reagent.
The green staining represents her one mRNA detected with a fluorescein labeled probe. And TSA fluorescein reagent nuclei stained with propidium. Iodide are colored blue in a double stained embryo.
Regions of distinct or overlapping expression can be unambiguously determined. And of this procedure reveals subcellular localization of mRNA as seen in these closeup views of her one staining. Active transcription of the gene is revealed by dots of staining in the nucleus.
Other cells exhibit cytoplasmic localization of the mRNA. In the double stained embryo. We can see cells transcribing both genes or either gene.
Separately, We've just shown you how to perform whole mount high resolution double fluorescent in situ hybridization of zebrafish embryos. When doing this procedure, it's important to remember that we first test our probes using standard alkaline phosphatase mediated staining probes that give a clean and strong signal will work well in fluorescent. In situ.
We have also successfully used immuno localization of beta catine in place of the propidium iodide staining. When we perform this variation, we do the hybridization reactions at 55 degrees Celsius instead of 65. So that's it.
Thanks for watching and good luck with your experiments.
