The overall goal of this procedure is to observe patterns of gene expression in zebrafish embryos during specific stages of development. This is accomplished by first hybridizing a di oxygen in labeled antisense rib probe to the MR mRNA of interest. The second step is to detect the ribo probe.
Using an antibody for doxygen in the antibody is conjugated to an alkaline phosphatase enzyme. The third step of the procedure is staining by the addition of NBT and BCIP, which react with the alkaline phosphatase enzyme to yield an insoluble purple precipitate. The final step of the procedure is to incubate sufficiently stained embryos in methanol to reduce non-specific staining.
Ultimately, results that show gene expression patterns in whole zebra fish embryos at specific stages of development can be obtained using whole mountain seed two hybridization. The main advantage of this technique over existing methods like traditional classroom demonstrations of histology or dissections, is that it gives students the opportunity to build conceptual bridges between molecular biology and trends in organismal development and evolution, thereby providing a truly integrative experience. Although these methods have traditionally been used to study the development of a few model organisms, they can easily be applied to other non-model organisms to understand the conservation of developmental pathways across vertebra taxa, as well as where these pathways deviate leading to evolutionary novelties.
This method may be unfamiliar to undergraduates, so some students may struggle at first, but learning this method will serve them well in undergraduate research experiences and upper division courses. To begin this procedure, use a transfer pipe to transfer around 50 zebrafish embryos into a micro refuse tube. Remove excess liquid and then pipette 1.5 milliliters of 4%para formaldehyde into the tube.
Place the tube of embryos on an agitator at room temperature overnight. The next morning, remove the fix and discard. Add 1.5 milliliters of phosphate buffered saline solution containing 0.1%tween 20 to the embryos to wash.
Use fine tipped forceps to manually coate the embryos if necessary, use a transfer pipette to place the coated embryos into a clean micro fused tube. Then pipette 1.5 milliliters of 25%methanol and PBST into the tube. After incubating for one hour, aspirate as much of this solution as possible without disturbing the embryos.
Then replace with 1.5 milliliters of 50%methanol in PBST and incubate for a minimum of one hour. Finally, replace with 1.5 milliliters of 100%methanol and place at minus 20 degrees Celsius. The embryos can be stored in methanol at minus 20 degrees Celsius.
When ready to use, pipette the methanol from the tube and discard pipette. 50%methanol in PBST into the tube and incubate for 10 minutes. Repeat this, wash a second time, then aspirate the solution and repeat the incubation with 25%methanol in PBST.
Finally, wash twice for 10 minutes each in 100%PBST prepare a 10%hydrogen peroxide solution in PBST. Immerse the zebrafish embryos in this solution at room temperature with the micro fued tube cap open for 10 to 20 minutes, depending on the age of the embryo. To remove any dark pigmentation using a transfer pipette, remove and discard the hydrogen peroxide solution.
Rinse the embryos in 1.5 milliliters of PBST multiple times to stop the reaction. Dilute a 50 milligram per milliliter stock solution of proteinase K one to 5, 000 in PBST. Typically one microliter of proteinase K in 4.999 milliliters of PBST.
Remove the PBST from the embryos. Using a transfer pipette discard and add 1.5 milliliters of proteinase K solution into the tube. Immerse the embryos in proteinase K solution at room temperature for three to 15 minutes, depending on the age of the embryos.
Wash with PBST multiple times to stop the reaction. Then remove the PBST and immerse the embryos in 4%power formaldehyde for 30 minutes of room temperature to re-fix, remove the para formaldehyde using a transfer pipette and discard. Add 1.5 milliliters of PBST to the embryos and wash for five minutes.
Carefully pipette the PBST from the vial and discard. Repeat the wash with a further two volumes of PBST. Then proceed to rip probe hybridization, prewarm water bath to 70 degrees Celsius and prepare the pre hybridization solution.
Then use a transfer pipette to remove and discard the PBST. Immerse the embryos in 0.5 milliliters of pre hybridization solution and incubate in the 70 degree Celsius water bath for two to three hours. Taking care not to disturb the embryos.
Pipette the pre hybridization solution from the micro refuse tube and discard. Gently pipette 0.5 milliliters of hybridization solution into the tube. Then quickly add previously synthesized doxygen in labeled ribo.
Probe into the tube ribo probe concentrations ranging around one microgram per milliliter are used. Incubate at 70 degrees Celsius overnight. The next day prepare solutions of 75%50%and 25%PHS in two times saline sodium citrate, as well as two times SSC and 0.2 times SSC solutions.
Warm more solutions in the 70 degree Celsius water bath. When the solutions have reached 70 degrees Celsius, retrieve them from the water bath. Then aspirate the hybridization solution from the tube and discard.
Add 0.5 milliliters of each warm P-H-S-S-S-C solution, incubating the embryos in each solution for 10 minutes at 70 degrees Celsius. Finally, incubate the embryos in the 0.2 times SSC solution at 68 degrees Celsius for 30 minutes. Aspirate the 0.2 times SSC solution from the tube and discard.
Incubate the embryos in 1.5 milliliters of malic acid buffer pH 7.5 at room temperature for 10 minutes. Repeat this, wash a second time. Prepare blocking solution according to the written protocol.
Transfer the embryos in MAB into each well of a 12. Well plate. Remove excess MAB and pipette one to two milliliters of blocking solution into each.
Well incubate at room temperature with agitation for at least three hours. Dilute the antigen in antibody one to 2000 in the remaining blocking solution. Incubate a room temperature for at least three hours with gentle vegetation.
Taking care not to disturb the embryos. Pipette the blocking solution from the wells of the plate and discard. Gently pipette the previously prepared anti doxygen in antibody and blocking solution into the wells incubate at four degrees Celsius overnight.
The next day, wash the embryos in five changes of 1.5 milliliters of malic acid buffer. Allow the embryos to incubate in malic acid buffer for five minutes. First, then perform buffer changes and incubate for two 10 minutes.
One 30 minutes and one 60 minute interval. Aspirate the final wash of meic acid buffer from the wells and discard pipette. 1.5 to two milliliters of alkaline phosphatase buffer into the wells and incubate for five minutes.
Repeat this process twice. Remove alkaline phosphatase buffer and add 1.5 to two milliliters of staining solution to the wells. Wrap the entire plate in aluminum foil to protect from light and gently agitate at 20 minute intervals.
Stop the agitation and observe the embryos under the dissecting microscope to check whether sufficient staining has been achieved. Once sufficient staining is visible, pipette the staining solution from the wells and discard. Replace with PBST to stop the staining reaction.
Repeat the washing process with another volume of PBST, pipette the PBST from the wells and discard to dehydrate the embryos Pipette 1.5 to two milliliters of 25%methanol in PBST into the wells and incubate for 10 minutes. Repeat the process twice with 50%methanol in PBST for a further 10 minutes each wash. Then transfer the embryos into 100%methanol and incubate for at least two hours to reduce non-specific staining.
Following incubation, pipette the methanol from the wells and discard to rehydrate the embryos. Wash twice with 50%methanol in PBST, followed by 25%methanol in PBST for 10 minutes each. Then washing 100%PBST twice for 10 minutes each time.
Finally, transfer stained embryos into 80%glycerol solution in PBST using a graded series of washes and store at four degrees Celsius until observation when performed correctly. Expression of the gene of interest is indicated by the appearance of a purple stain on the zebra fish embryo. This image shows a zebra fish embryo 24 hours post personalization, which has been hybridized with RIBA probes specific for AAL DH one A two specific staining can be seen in the eyes, hind brain, pectoral, fin bud, ria, and somites.
This image shows a zebra zebrafish embryo at the 13 somites stage of development that has been hybridized with a probe specific for FGF eight A.In this case. Specific staining is seen in the tline kelon, dorsal Dion, Kelon midbrain, hind brain boundary, somites, and tail bud. This image shows a 22 hours post fertilization zebra fish embryo that has been hybridized with the probe specific for PAX two A.A robust marker useful for visualizing the nervous system.
Specific staining can be seen in the choroid fissure, midbrain, hind brain boundary, otic vesicle, and spinal cord neurons Once mastered. This technique can be modified to be performed across several laboratory sessions depending on the course schedule. Time and effort outside of the classroom may be required by the instructor.
After watching this video, we hope that you're more comfortable with the idea of bringing whole mount and C two hybridization into an undergraduate laboratory. As students develop these skills, knowing about this technique can facilitate their transition to upper division or graduate research experience.
