1. The same as for Subheading 7.2. (although with different freezing medium).
2. Freezing medium VS1: PBS with Ca2+ and Mg2+ containing 0.4% BSA (Sigma A4161), 20% (v/v) DMSO (Sigma D2650), 15.5% (w/v) acetamide (Sigma A0500), 10% (v/v) propylene glycol (Sigma 1009), 6% (w/v) polyethylene glycol 8000 (Sigma P2139).
3. Freezing medium VS2: PBS with Ca2+ and Mg2+ containing 0.4% BSA and 12.5% (v/v) freezing medium VS1.
4. Freezing medium VS3: PBS with Ca2+ and Mg2+ containing 0.4°/O BSA and 25% (v/v) freezing medium VS1.
5. Freezing medium VS4: PBS with Ca2+ and Mg2+ containing 0.4% BSA and 50% (v/v) freezing medium VS1.
16.4.2. Methods 126.96.36.199. Freezing Embryos
1. Prepare one 35-mm tissue-culture dish containing VS1 and chill at 4°C. Prepare another 35-mm tissue-culture dish containing VS2 and with VS3. Incubate at room temperature.
2. Place the rat blastocysts (5 d post-hCG) in the dish containing VS2, and incubate for 5 min at room temperature.
3. Transfer the embryos to a dish containing VS3, and incubate at room temperature for 5 min.
4. Wash the embryos in the chilled (4°C) dish of VS1. Transfer the embryos in a volume of 40 ||L to a precooled 4°C polypropylene tube or freezing straw. Chill at 4°C or 15 min.
5. Plunge the tube into liquid nitrogen and store.
188.8.131.52. Thawing Embryos
1. Chill freezing media VS4 and VS3 to 4°C.
2. Prepare one 35-mm tissue-culture dish containing VS2 and two dishes containing 0.4% BSA in PBS with Ca2+ and Mg2+. Incubate all three dishes at room temperature. Prepare two 35-mm dishes containing M16, and incubate in a 37°C incubator pregassed at 5% CO2.
3. Thaw the tubes on ice.
4. Add 200 |L of chilled VS4 to the thawed tube, and incubate at 4°C for 10 min.
5. Add 400 |L of chilled VS3, and leave at 4°C for 10 min.
6. Transfer the embryos to the dish containing VS2, and maintain at room temperature for 5 min.
7. Wash the embryo twice in the dishes of 0.4% BSA in PBS with Ca2+ and Mg2+.
8. Transfer the embryos into pseudopregnant mothers.
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