After watching Eric and Mariana sacrifice a male frog for his testis and use them to fertilize the eggs laid by the female frogs, Toby, Dahee, and I started to fix frog embryos for In Situ Hybridization by ourselves. The first step is to fix the embryos at different stages in glass vials with MEMFA solution at room temperature at 4 degrees Celsius overnight. Afterwards, change the embryos to fresh MEMFA after the first 30 min, using an end-over-end rotator. MEMFA Recipe is as follows: 1/10 10x MEM, 1/10 Formaldehyde, 8/10 Deionized water. The second step is to change the embryos into PBST three times, for five minutes each, using the rotator. The third step is a Proteinase K treatment for embryos with vitelline membrane, with 10 micro grams per milliliter in PTW (1:2000 dilution) for 5 minutes. The fourth step is to wash the embryos in PBST twice, for 5 minutes each, using the rotator. The second, third, and fourth steps can be skipped if the vitelline membrane has been removed, which usually occurs in embryos at stage 22 or beyond. The fifth step is to change the embryos into 100% ethanol, then leaving them on the end-over-end rotator for 20 minutes. Then changing them into fresh ethanol and leaving them on the end-over-end rotator for 30 minutes. The sixth step is to replace the ethanol with 100% methanol by washing the embryos twice, for 30 minutes each, on the rotator. Afterwards, the embryos need to be stored in methanol in the -20 degrees Celsius freezer. The frog embryos that we fixed were at Stages 9, 18, and 22. Overall, this protocol is extremely straightforward, but it requires time and patience, due to the large wait in between the washes.
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Prachi MishraOh hi there! ArchivesCategories |