The overall goal of this procedure is to define gene expression patterns in whole mouse embryos. This is accomplished by first designing and generating a specific ribo probe for the gene of interest. Next, the embryos are collected and treated for hybridization.
Then the labeled probe is hybridized to the embryos. The final step is to visualize the hybridized probe by immunohistochemical detection. Ultimately, results can be obtained that show gene expression patterns in whole embryos or embryonic sections.
After imaging with a stereos, dissecting microscope, or a regular brightfield microscope, Generally individuals new to this method will struggle because the institute hybridization procedures are lengthy and technically demanding, there are multiple important steps that collectively contribute to the quality of the final result. First, generate template DNA by amplifying the sequence of interest using gene specific PCR primers designed with farge transcription promoter sequences on the five prime ends. Then purify the PCR product and include a proteinase K digestion step according to the written protocol.
Then set up an in vitro transcription reaction and incubator optimal temperature for two hours. After DNA's digestion of the DNA template, purify the ribo probe using column purification, estimate probe yield by using a NanoDrop or microplate absorption reader. Then adjust the probe concentration to 100 nanograms per microliter according to the estimated yield.
Then check the quality of the probe by denaturing acrylamide gel electrophoresis as outlined in the written protocol and by measuring the specific activity of the RIBA probe, a good probe should yield a single discrete band on the gel with minimal smear to measure specific activity, perform serial dilution of the ribo probe to obtain a range of dilutions from 10 to the minus two to 10 to the minus five. Then pipette one microliter of each dilution onto an 82 millimeter diameter piece of high bond n plus membrane, along with one microliter of an unlabeled DNA sample as a control. Crosslink the filter immediately in a UV crosslinker at 125 millijoules after crosslinking.
Add 10 milliliters of 1%blocking reagent in one times TN buffer incubate room temperature for 30 minutes after blocking is complete. Remove the blocking solution and wash the filter for 15 minutes at room temperature in 10 milliliters of one times TN buffer while washing the membrane. Prepare a one to 5, 000 dilution of anti doxygen in FAB fragment in 10 milliliters of one times TN buffer after washing.
Incubate the membrane in this solution for 30 minutes Following incubation, wash the membrane twice for 15 minutes each in 10 milliliters of one times TN buffer. After the final wash, aspirate the wash solution from the Petri dish and replace with five milliliters of BM purple substrate. Incubate the membrane in this solution for 30 minutes.
After 30 minutes have elapsed, check the membrane for the presence of a visible signal emitted from the spot corresponding to the 10 to the minus four dilution. If the signal is visible, stop the color reaction by washing with five times TE twice for five minutes each time. If no signal is visible after 30 minutes, the probe should be discarded.
Collect mouse embryos in ice cold PBST. Then sort the embryos according to genotype into four milliliter screw capped files containing PBST. Fix the embryos with 4%PARALDEHYDE in PBST unless otherwise specified.
All incubation and wash steps are done with solutions filled to the lower level of the vial neck, which is about four milliliters in volume. Leave the embryos overnight at four degrees Celsius after fixation. Aspirate the 4%paraldehyde in PBST with a glass pasta pipette and replace with PBST.
Place the vial on ice for five minutes to wash. Repeat this process to wash the tissue for a second time. Next, aspirate the PBST and dehydrate the embryos stepwise through a methanol in PBST series into 100%methanol.
Note that the pipette tip is kept on the opposite side of the vial to avoid damage to the embryos if desired, the embryos can be stored at minus 20 degrees Celsius in 100%methanol for up to eight months and maybe longer until use. Transfer the embryos to a dish, puncture the heart and head of E 10.5 and older embryos with a microdissection knife while the embryos are still in methanol. This allows wash solutions to enter the brain ventricles and heart chambers and reduces background staining.
Then rehydrate the embryos by successive washes in a methanol in PBST series. Finishing with two washes for five minutes each in 100%PBST bleach the embryos by preparing a solution of four parts PBST to one part 30%hydrogen peroxide and incubating the embryos in this solution for one hour on ice. During the incubation, place the freshly prepared hybridization buffer at 65 degrees Celsius to prewarm.
Wash the embryos with PBST twice for five minutes each. During the final wash, make a 20 milligrams per milliliter proteinase K stock with DEPC water. Then serially dilute the stock to make 10 micrograms per milliliter.
Proteinase K in PBST aspirate the final wash solution and replace with one milliliter of the 10 microgram per milliliter proteinase K solution. Place the vial vertically in a tube rack within a 25 to degrees Celsius water bath and incubate for 10 to 30 minutes, depending on the size of the embryos. Make a note of the start time as optimal and replicable.
Incubation time is critical to enable penetration of the ribo probe without over digestion of the embryo. To stop the protease K digestion aspirate the proteinase K solution 30 seconds before the incubation time has elapsed. Then when the incubation time elapses add freshly made two milligrams per milliliter glycine in PBST.
Let's stand for five minutes at room temperature. Then repeat the glycine wash for another five minutes. Then wash twice in PBST for five minutes each.
Next refix the proteinase K treated embryos by immersing the embryos in 4%PARALDEHYDE in PBST supplemented with 0.2%glutaraldehyde for 20 minutes. Then wash the embryos in PBST three times for five minutes each. After the last wash, aspirate the PBST and replace with one milliliter of prewarm hybridization buffer.
Once the embryos have settled, aspirate the hybridization buffer and replace with one milliliter of fresh prewarm hybridization.Buffer. Incubate the vials vertically in a 65 degrees Celsius oven with a shaking platform for one hour. Prepare hybridization buffer containing 0.5 micrograms per milliliter of labeled ribo.
Probe for embryos up to E 8.5 or 0.1 to one microgram per milliliter of probe for embryos past E 8.5. Following the incubation, replace the pre hybridization solution with 0.4 milliliters of this solution and hybridize overnight at 65 degrees Celsius. The next day, check the quality of the embryos.
If the embryos have been overtreated with proteinase K, they may stick to the size of the vial, will appear extremely transparent or fall apart. Embryos that appear this way should be discarded as processing them further will not result in interpretable data. Replace the hybridization buffer with four milliliters of solution.
One at 70 degrees Celsius. Maintain this temperature and twice for 30 minutes each for E 8.5 embryos or three times for 30 minutes per wash. For E 9.5 and older embryos during the last wash, prepare four milliliters of 50%solution, one 50%solution two for each vial and prewarm to 70 degrees Celsius after the last wash.
Incubate the embryos in this solution for 10 minutes, then wash three times for five minutes per wash with four milliliters of solution. Two at room temperature with rocking after washing. Replace the solution with fresh solution.
Two containing 100 micrograms per milliliter RNAs A and 100 units per milliliter RNAs T one incubate at 37 degrees Celsius twice for 30 minutes per wash. Next washing solution three at 65 degrees Celsius with rocking twice for 30 minutes each wash for E 8.5, three times for 30 minutes per wash for E 9.5 and older. Finally, wash three times for five minutes per wash with one times TBST.
Then proceed to antibody detection. Aspirate the final TBST wash from the vial and replace with 0.5 milliliters of one times TBST plus 10%heat treated sheep serum. Place the vial vertically on a platform shaker at room temperature and incubate with rocking for at least two and a half hours to block non-specific antibody binding sites.
After the incubation time has elapsed, aspirate the blocking solution and replace with fresh one times TBST plus 10%serum containing a one to 2000 to one to 5, 000 dilution of subtracted alkaline phosphatase, conjugated sheep antigen, and FAB fragments antibody and incubate overnight at four degrees Celsius. Wash three times for five minutes each with one times TBST. Then five to six times for one hour reach at room temperature and finally overnight at four degrees Celsius to remove excess antibody.
The next day. Wash the embryos twice for 20 minutes per wash with freshly made alkaline phosphatase buffer. This is an important procedure to reduce background.
Replace the last wash with one milliliter of prewarm BM purple AP substrate. Incubate at room temperature while protected from light and watch for signal periodically via dissecting microscope signal. For abundant messenger.
RNA should be visible within 15 minutes. When the signal is clearly visible or if background staining begins to be a problem, stop the reaction by washing twice for five minutes per wash with one times PBST plus five millimolar EDTA capture images to document the staining pattern of the whole mount embryo. Once this has been done, return the embryos to a vial of BM purple substrate and incubate for 16 to 24 hours at room temperature until the external surface of the embryo becomes dark blue to allow the stain to penetrate throughout the embryo.
Then transfer the embryos to 4%parmal hide in PBST to refix for several hours or overnight at four degrees Celsius. This final fixation step is crucial for long-term preservation of the staining pattern. Finally, proceed to paraffin, embedding and sectioning according to the procedures outlined in the written protocol to reveal this staining.
This image shows a successful hole mount in C two hybridization of an E 10.5 embryo using a gata three specific ribo probe. Here is an image showing an E 11.5 embryo that has been successfully labeled with a Fox G one specific ribo probe. This image shows high background and weak signal from a failed in C two hybridization with a partially degraded gata three probe illustrating the importance of performing careful quality control of probes to ensure high quality results.
Here is an image of another unsuccessful procedure. In this case, the procedure was performed without puncturing the head resulting in a high level of background staining in the ventricles as indicated by the arrow heads. After watching this video, you should have a good understanding of how to perform high quality Institute hybridization to define gene expression patterns on mouse embryos for spatial details and the temporal or genotype comparisons.
