Visualization of dead cells with Acridine Orange

Owned by callol
Last updated: Nov 12, 2009Version comment
1 min read

1. Embryo manipulation.

Embryos of all stages can be used and do not need any special manipulation. If considered necessary, PTU treatment can be applied to reduce pigmentation after 24 hours post fertilization.

2. Acridine Orange preparation.

Acridine Orange (Sigma A6014) is prepared at 1mg/ml (100X) in milliQ water and stored at -20C (light protected).

3. Incubation.

Incubate alive embryos at room temperature with Acridine Orange 1X during 30 minutes in E3 medium.

Wash 3 times during 10 minutes (shacking is optional).

4. Visualization.

Treat embryos with tricaine or an equivalent anesthetic.

Visualize dead cells under a dissecting microscope with a green fluorescent filter. We use a a filter with excitation 470/40. 

Methyllcellulose (MC) can be used to position the embryos. Attention, we have noticed MC might increase the apoptotic signal or induce new signals after a short period of time.

GOOD LUCK!