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  • Bruno Vellutini 23:45 on 2013/08/12 Permalink
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    Membranipora inhibitor time points 2-12h 

    Repeated the spawning procedure from last time. Just cut a piece of kelp from the flowing tank and add to a bowl with UVFSW and put under the scope with direct light. Had 5 bowls of embryos!

    Since I found that 10 µM is a good concentration for a mild phenotype, I’m repeating the experiment with DMSO control and 10 µM in 500 µL at 10 °C.

    CONTROLDMSO 1:500A: 10h30 U0126 10 µM1:1000A: 10h30
    I: 12h30 (2h) 2-4 cell
    I: 14h30 (4h) 4-8 cell
    I: 16h30 (6h) 8-16 cell
    I: 18h30 (8h) 16-32 cell
    I: 20h30 (10h) 32-64 cell
    I: 22h30 (12h) +64 cell

    I added the inhibitor at 2h, 4h, 6h, 8h, 10h, 12h post-activation.

    Also set a 4D with 10 µM embryo and the recording was good, but condensation shifted focus overnight.

    13/08/13

    Checked the embryos and they look ok. It is not easy to differentiate them at this stage.

    14/08/13

    Now it is easy to differentiate embryos. Many treated ones are radialized and swimming. There are also many larvae that look shortened and wider. I may need to repeat this batch since I did not control well the volume I added when picking, thus most likely on time point 2h it was less than 10 µM, which might explain the number of larvae (different from timepoint 4h where almost no larvae were present).

    15/08/13

    Fixed all samples (3d).

     
  • Bruno Vellutini 14:19 on 2013/08/12 Permalink
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    4D Membranipora U0126 treatment 10 °C 

    Folder: BCV_Mm_2013_08_12

    First 4D. Started in the morning. Water from condensation invaded the oil during overnight, after gastrulation. I think the tracing is fine since the embryo had already been disrupted, so should be useful.

     
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