This procedure begins with fixation, methanol, and proteinase K treatment of the embryos, followed by overnight incubation with probe one diggen in or dig and probe. Two fluorescein isothiocyanate or ZI embryos are incubated with dig antibody following color reaction with dig alkaline phosphatase. Activity is inactivated and embryos are incubated.
With FSE antibody color reaction is performed with Zi and embryos are fixed. Hi, I am Delphine from the lab of Rick Finnel at the Center for Environmental and Genomic Medicine at Texas a and m University in Houston. Today we will show you procedure for performing two colorant situ hybridization in the chick embryo.
We use this procedure in the lab to analyze the effects of antiepileptic drugs on gastric and neural genes. So let's get started To perform in situ hybridization on chick embryos, first open an egg by tapping the shell with forceps and removing pieces of the shell. Remove the thick albumin with forceps and tilt the yolk sack with coarse forceps so that the embryo faces upwards using fine scissors.
Cut a square of yolk sack around the embryo. Remove the embryo from the yolk with a spoon and place in a dish containing ice. Cold dey PBS under a dissection microscope Carefully remove the membranes and ylk and transfer the embryo to a fresh dish containing ice.
Cold dey PBS hold the embryo down with forceps and aspirate the dey PBS in the fume hood. Replace this with 4%paraform aldehyde in dey PBS and allow the embryo to fix at four degrees Celsius overnight. After the embryos are fixed, aspirate the fixative solution and dispose properly as chemical waste.
Fill the dish with ice cold dey PBS using a blunt end micro capillary needle or a microdissection knife perforate the head and the heart cavities. To prevent trapping of probe. Place the embryos in DEP CPBS containing 0.1%tween 20, then dehydrate the embryos in a methanol series wash for 10 minutes each in 25%50%75%and 100%methanol in PBT.
Repeat the 100%methanol wash. The embryos may now be stored at minus 20 degrees Celsius in methanol. In 20 milliliter glass scintillation vials.
To perform the insitu hybridization, it will be necessary to first rehydrate the embryos. To do this, wash the embryos 10 minutes each in 75%50%and 25%methanol in PBT. Finally, wash twice for 10 minutes each.
In PBT, remove the PBT solution from the vials and replace with a proteinase K solution of five micrograms per milliliter. Three milliliters per vial. Gently rolled the vial to make sure the entire contents are exposed to the proteinase K solution.
Leave in proteinase K solution for one to five minutes depending on embryo stage. Meanwhile, prepare ice cold fixative solution using an RNA free past pipette, remove the proteinase K solution and add two milliliters of the fixative solution. Immediately place the vial on ice for exactly 20 minutes.
Next, wash the embryos at room temperature on a mutator. Perform two 10 minute washes in PBT followed by one 10 minute wash in 50%hybridization buffer in PBT. Then wash once in 100%hybridization buffer incubate the embryos in two milliliters of hybridization buffer for four to five hours.
At 65 degrees Celsius. The embryos are now ready to be hybridized with probes dig and fitzy coupled probe synthesis is described in the supporting material. The probe expected to give a strong signal should be synthesized with FSE containing nucleotides, whereas the weaker probes should be synthesized with dig containing nucleotides.
Pre-warn probes in hybridization buffer using a two milliliter vial. Remove the hybridization buffer from the embryos and promptly replace the hybridization buffer with the probe solution. It is important to not let the embryos dry incubate for 16 hours at 65 degrees Celsius.
After the probe hybridization step, replace the probe solution with hybridization buffer. Perform two 30 minute washes at 65 degrees Celsius, then wash for 15 minutes in a one-to-one mixture of hybridization, buffer and maleic acid buffer again at 65 degrees Celsius. After this step, you no longer need to use RNAs free conditions.
Now you are ready to perform the dig color reaction followed by the Fitz color reaction. To perform the antibody hybridization and color reaction first, wash the embryos twice for 20 minutes each in maleic acid buffer. Then wash for one hour in 2%VBR in maleic acid buffer.
Finally, watch for five hours in blocking buffer Dilute dig antibody one to 2000 in blocking buffer and incubate the embryos in this solution on a mutator overnight at four degrees Celsius. The next day perform three 10 minute washes in maleic acid buffer followed by three one hour washes in maleic acid buffer. Wash twice for 20 minutes each.
In NTMT add 200 microliters N-B-T-B-C-I-P substrate to 10 milliliters. NTMT immediately remove NTMT from the vial with the embryo and replace with one to two milliliters. N-B-T-B-C-I-P buffer place in the dark.
Monitor the color reaction after 20 minutes and at 20 minute intervals thereafter. When the blue color has developed appropriately, wash the embryos in PBS for 10 minutes. Then replace the PBS with 4%paraform aldehyde in PBS.
Fix overnight at four degrees Celsius. Transfer the embryo to a new syn installation vial wash in PBT twice for 10 minutes each, followed by two 10 minute washes in maleic acid buffer. Incubate the embryos in maleic acid buffer for 30 minutes at 65 degrees Celsius.
Now it is time to perform the second antibody incubation and color reaction. In order to detect our second fits e labeled probe, wash the embryos twice for 10 minutes each in maleic acid buffer. One hour in 2%BBR in maleic acid buffer.
And then five hours in blocking buffer dilute the alkaline phosphatase coupled anti FTSE antibody one to 500 in blocking buffer and incubate the embryos in this solution overnight at four degrees Celsius. The next morning performed three 10 minute washes in maleic acid buffer followed by three one hour washes in maleic acid buffer. Next, wash twice for 20 minutes each.
In Tris HCL, prepare five milliliters of vector red working solution in tris HCL According to the manufacturer's protocol. Immediately remove the triss HCL from the vial and replace with the vector red buffer. Place the vials in the dark.
Monitor the color reaction after 30 minutes and at 30 minute intervals thereafter, after the red color has developed, transfer the embryos to PBS. You are finally ready to fix and process the embryos. Transfer them to a fixation dish containing 4%paraform, aldehyde and fix for 20 minutes at room temperature or overnight at four degrees Celsius.
Wash twice in PBS 10 minutes per wash to process for photography. Transfer the embryos to 20%glycerol in PBS. This will make the embryos translucent to process for wax sectioning.
Transfer the embryos back to PBS wash twice for 10 minutes each dehydrate in a methanol series for 10 minutes each replace the methanol with propanol and incubate for three minutes. Remove the propanol and replace with 1 2 3 4 tetra hydro nap filine for 20 minutes, followed by histo clear, two washes for one hour each. Replace with a one-to-one mixture of histo clear and wax and incubate at 60 degrees Celsius for one hour.
Replace this solution with wax and process for histology. This embryo is labeled with a digo oxygen in socks, two probe in blue and a fitzy delta one probe which appears in red. The first color reaction reveals the expression pattern of socks.Two.
A marker for the neural plate developed in the laboratory of Dr.Robin level badge sox two positive cells appear blue following reaction with N-B-T-B-C-I-P. The same embryo is then processed for the second color reaction, which reveals the expression pattern of delta one. A marker for the segmental plate developed in the laboratory of Dr.Domingo Enrique Delta one positive cells appear red, following reaction with vector red.
This embryo is labeled with a digo in PAC six probe in blue and a fitzy SHH probe in red. The first color reaction reveals the expression pattern of PAC six, A marker for the neural folds and developing somites developed in the laboratory of Dr.Martin Goulding Pac. Six positive cells appear blue following reaction with N-B-T-B-C-I-P.
The second color reaction reveals the expression pattern of SHHA marker for the Noor and ventral midline of the neural tube developed in the laboratory of Dr.Cliff Tabban. SHH positive cells appear red, following reaction with vector red. We've just shown you how to perform to color and c hybridization in the chicken ry.
When performing this procedure, it's important to remember to use depth C treated solutions as well as autoclave, pfas, and sterile vi. In order to minimize contact with RNAs is in steps three to five. So that's it.
Thanks for watching and good luck with your experiments.
