Method for preparing villous chromosomes

Method for preparing villous chromosomes

The villi are derived from the mesoderm of the embryo. The earliest primary dry villi appear at the beginning of the third week of gestation, and about 8 weeks of gestation is the most vigorous period of villi development. At present, the fluff materials at home and abroad are mostly selected in 8-9 weeks. Research data show that villus sampling does not affect embryonic development and placental function. But sampling failure can lead to embryo loss and termination of pregnancy.
The former should first check vaginal secretions to determine the cleanliness of the vagina and prevent intrauterine infection caused by sampling. The second is to do B-ultrasound examination, one is to determine the size of the embryo, the second is to determine whether the embryo has cardiovascular pulsation, and the third is to determine the position of the embryo in the uterus to determine the time of sampling, whether to take the sample, and the angle and depth of the sampler entering the uterus.
1. Experimental materials
Gynecological vaginal washes, villus sampler, 5ml syringe, petri dish, tweezers,  formic acid, sodium citrate, glacial acetic acid, PMRI 1640, colchicine, microscope glass slides, etc.
2. Preparation of culture medium
PRMI 1640 10-15ml
Colchicine 10ug/ml 1ml
3. Operation process
(1) Rinse the vagina with 1‰ xinjie and sterilization, fix the cervix with oval forceps, select the angle and concentration according to the prompts of gynecological examination and B-ultrasound, and insert the villus sampler exploratoryly. At this time, take out the inner core of the sampler, connect a 5ml syringe, and draw out 4-5ml. At this time, bloody liquid can be seen flowing into the syringe;
(2) Take a clean petri dish and add 5ml of PRNI 1640, containing 0.06ug/ml of colchicine. Slowly withdraw the sampler, inject the aspirated material into the petri dish, rinse the syringe and sampler with 1640, and mix well. Set the incubator for 30-40 minutes;Petri Dish Sterile,Petri Dish 90mm Sterile,Disposable Petri Dish

(3) Select well-developed villi and put them into another small petri dish, and use 0.075M pre-warmed at 37°C. KCL was rinsed twice with a 1:1 mixture of 10% sodium citrate.Petri Dish Sterile,Petri Dish 90mm Sterile,Disposable Petri Dish
(4) Cut the fluff with small ophthalmic scissors, and then add 2 drops of collicine to rinse the hypotonic solution for 30 minutes on the way;
(5) Add 0.2 ml of 3:2 methanol glacial acetic acid fixative solution, pre-fix, 1000rpm for 8 minutes, discard the supernatant;
(6) Add 3ml of freshly prepared 3:2 fixative solution; fix for 30 minutes, 2000rpm for 8 minutes, discard the supernatant;
(7) For the second fixation, add 3ml of 3:1 methanol glacial acetic acid fixative solution for 30 minutes, 2000rpm for 8 minutes, discard the supernatant;
(8) After centrifugation, add the remaining volume of 60% glacial acetic acid, mix well, add the same amount of methanol after 2 minutes, mix well, 2000 rpm for 8 minutes, discard the supernatant.

(9) 3ml of 3:1 formazan fixative overnight, and ice-water to make microscope glass slides;
(10) Test the film at 80°C for 2 hours, and cool it naturally.
(11) G-banding treatment, observe the microscope glass slide.

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