Membranipora staining with Phalloidin 647 and Sytox Green in TDE


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I scanned the U0126 treated embryos with DAPI+Phallacidin in glycerol and the outcome was far from optimal. Penetrance was not great and DAPI is always week… So I decided to try to get a nice morphological staining using TDE, to scan the whole sample without loss; Sytox Green, for strong nuclear staining; and Phalloidin 647, for cell linings and actin. I think this should give a clear picture and nice composition for a morphological stance.

I selected different stages from cleavage to cyphonautes and added to 2 wells. In one I’m doing the staining above and in the other I’m just mounting them in TDE without phalloidin or sytox. I want to clarify if they have auto-fluorescence at all when embedded in TDE. I suspect they have it on the green.

Phalloidin + Sytox Nothing, just unstained embryos

I got 6.25 µL of Phalloidin 647, dried in a tube and diluted in 250 µL of 0.1% PTx with 1:1000 dilution of Sytox Green. Incubated covered for 1h40 and washed 4x 5 min in PBS. Kept the well in the dark at 4 °C.

11/04/14

Embed samples in TDE and mount slides to check signal.

12/04/14

Confocal?