Cell number counting in preimplantation embryos 胚胎植入前细胞计数

Introduction
The following is a simple procedure for determining cell number in preimplantation embryos. Note that DAPI has higher photostability than Hoechst 33342.

Other DNA-binding dyes can also be used including propidium iodide and ethidium bromide. Staining with propidium iodide is a little more complicated because the dye also stains RNA and an RNAse step is often added. The Molecular Probes website has much useful information on the properties of various DNA binding dyes.

Preparation of Embryos
1. Remove embryos from embryo culture medium and wash 2 times in 100 µl drop of PBS containing 1 mg/ml polyvinylpyrrolidone (PVP) by transferring the embryos from drop to drop.

2. (Optional) Fix embryos in 100 µl drop of paraformaldehyde solution [4% (w/v) in PBS, pH 7.4] for 1 h at room temperature. Wash the embryos 3 times in 100 µl drop PBS/ PVP by transfering the embryos from drop to drop.

Preparation of Hoechst 33342 dye
(all solutions are made in light-proof tubes - wrapping in aluminum foil is sufficient)

Stock 1: Dilute 25 mg of Hoechst 33342 (Sigma B2261) in 2.5 ml of distilled water. The concentration of this solution is 10 mg/ml. Store at 4 C.

Stock 2: On the day of use, dilute 5 µl of Stock 1 solution in 10 ml of PBS containing 1 mg/ml polyvinylpyrrolidone. The concentration is 5 µg/ml.

Working Solution: Dilute 200 µl of the Stock 2 solution in 800 µl of PBS-PVP for a final concentration of 1 µg/ml.

Preparation of DAPI
(all solutions are made in light-proof tubes - wrapping in aluminum foil is sufficient)

Stock solution: Dissolve 0.1 mg DAPI in 10 ml deionized water, saline, PBS or dimethylformadide. Store at 4 C for 6 months. Note: It takes several hours for DAPI to dissolve into solution.

Working solution: On the day of use, add 100 µl stock solution to 900 µl PBS/PVP. Use once and discard.

Staining the Embryo (this step should be performed with the lights dimmed)
1. Transfer embryos to a 50 µl microdrop of working solution.

2. Stain the cells for 10 minutes.

3. Wash embryos two times in 100 μl drops of PBS-PVP transferring the embryos drop to drop.

Mounting Embryos to Slides
1. Prepare clean microscope slides by dipping in a 1:10 poly-l-lysine solution (Sigma P8920) for 2 minutes. Allow the slides to dry.

2. Transfer the embryos to a poly-l-lysine coated slide in a minimal volume and allow embryos to dry for 15 minutes at room temperature. Use a diamond pen to scratch a circle on the bottom of the slide around the area where the embryo is located.