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  • Bruno Vellutini 19:30 on 2014/04/10 Permalink
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    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?

     
  • Bruno Vellutini 18:47 on 2014/04/10 Permalink
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    Pilidium larvae of Lineus longissimus look wonderful and happy. Annie changed 1/3 of the water today and added more food. There was algae sticking to the bottom, so maybe next time it needs to be scrubbed so that it gets exchanged.

    I’ll do the next cleaning on Monday.

     
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