(Idea for tuning Blog gun Shora )
Friends!
Today we will learn how to freeze mouse embryos, in a fast, (ultrarapid freezing), simple and for the whole family.
This system has the advantage of being faster than conventional freezing (10 minutes instead of two hours) and also avoids having to have a biocongelador. The main drawback is that it can only be used in relatively small cells, so often used to compacted morula stage, ie in embryos obtained on day 3 in the afternoon, considering day 1 on the appearance of vaginal plug, which is pregnancy diagnosis used in mice: The semen of the male has a sticky last fraction compact forming a plug in the vagina of the female, which may be visible to the morning after intercourse.
need the following materials:
Females Males
mouse
mouse HCG (human chorionic gonadotropin)
PMSG (equine chorionic gonadotropin)
Alcohol burner Paper towel
90% ethanol surgical material (normal scissors fine point, two-tooth forceps and two needle nose pliers)
binocular magnifier glass pasteur pipettes
siliconized tube system to handle the embryos into the pipette. Petri plates
Middle
M2 35mm tempered in hot plate at 37 ° C
fine hypodermic needle (no.)
Hyaluronidase. Middle
freezing. Middle
defrost Straws
Empajuelador
Bank of liquid nitrogen nitrogen insulated box Rack
liquid metal foil thickness 2mm
STEP 1: FOR EMBRYO SUPEROVULATION in morula stage animalario
-Hours: The light comes on at 7:00 and off at 21:00. This step is critical for coupling the daily hormonal rhythm of mice to which we are going to impose.
-Intraperitoneal injection of PMSG at 15:00 of the day-
-2 intraperitoneal injection of HCG at 15:00 on day 0
-are placed with males in the day 0, popping the cap 1 day ago
-sacrifice of the females on day 3 from 17:00. The embryos are at uterotubal union, so it will be necessary washed oviduct and uterine horn on each side.
STEP 2: PREPARATION OF THE MEANS OF FREEZING
"He uses the same means by which embryos are collected (usually M2). Penetrating cryoprotectants are added (low molecular weight alcohols such as glycerol or ethylene glycol) and non-penetrating (macromolecular lining the embryo and help your dehydration, such as sucrose, bovine serum albumin or fetal calf serum.
- An example would be, M2:
• 5% fetal bovine
· 0.25 M sucrose
· 3M ethylene glycol.
-is sterilized by filtration, and before use must be shaking, because the ethylene glycol has a tendency , to choose and stick to the walls of the bottle. Is kept in the refrigerator for a month, although it is better to have the shortest possible time.
STEP 3: PREPARING THE MIDDLE OF THAWING
"It's PBS with 5% fetal bovine serum, but in this case takes 0.5 M sucrose. Receive the same treatment and behaves like that of freezing.
STEP 4: METHOD OF FREEZING
few minutes before starting the procedure, place a sheet of aluminum of about 2mm in thickness in a polystyrene box filled with liquid nitrogen so that the distance between the surface of nitrogen and the plate is 50mm, and is held in a to stay fully horizontal. (With a rack or similar method)
Place a drop of about 75 ul of freezing it on a plate, and it was pipetted into the smallest possible volume 10-15 embryos. From that moment must have 5 minutes. During that time, to gently agitate the embryos. These must be incurred due to the hyperosmotic solution and float, and later (in about 3 min) fall to the bottom, at which point you can start empajuelar.
straw was loaded into a drop of medium without embryos as 15mm, 10mm then air, then drop half with embryos air again, and a half to reach the end of the straw. Open end is sealed with heated tweezers, and when they have met the 5 minutes since the embryos entered the freezing medium, place the straw in the iron on nitrogen.
Straws 2 minutes should remain on the plate, and then immersed in liquid nitrogen. From here, you can take to the bank and stay long periods of time.
STEP 5: METHOD OF THAWING:
a) Bring the thawing medium, and pour in 2 ml PBS.
b) Remove the straws from liquid nitrogen 20 seconds and let the vapors evaporate so it does not explode when heated.
c) Browse to the bathroom to 37 ยบ for another 20 seconds
d) Dry the straw, cut the seal, resting on the thawing medium, where embryos must remain at least 5 minutes, stirring gently.
e) Wash in M2
f) To plate with droplets of KSOM * whether to grow or transfer immediately whether to do the latter, it increases the success of the operation.
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