Tagged: Lineus viridis Toggle Comment Threads | Keyboard Shortcuts

  • Bruno Vellutini 11:45 on 2013/05/24 Permalink
    Tags: Lineus viridis   


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    Selected remaining juveniles ~2 month and incubated with 20 µM EdU from Click-iT Kit for 3h (stock concentration is 10mM, 2µL/1000µL). Relaxed with MgCl2 for 30min in cold room and fixed with 4% PFA for 1h in cold room + 1h at RT. Permeabilized with PBS+Triton-X and stored at 4 °C in PBS.

     
  • Bruno Vellutini 20:47 on 2013/05/07 Permalink
    Tags: Lineus viridis   

    Selected 12 juveniles ~1 month and incubated with… 


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    Selected 12 juveniles ~1 month and incubated with 20 µM EdU from Click-iT Kit for 3h (stock concentration is 10mM, 2µL/1000µL). Relaxed with MgCl2 for 30min in cold room and fixed with 4% PFA for 1h in cold room + 1h at RT. Permeabilized with PBS+Triton-X and stored at 4 °C in PBS.

     
  • Bruno Vellutini 12:54 on 2013/05/06 Permalink
    Tags: Lineus viridis   

    Fixing 32d Lineus viridis juveniles 


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    After failing to properly relax juveniles yesterday, I did the following.

    1. 45 min in MgCl2 in cold room
    2. 2 min in %2 cystein diluted in MgCl2
    3. 2 washes with MgCl2
    4. 15 min in MgCl2 in cold room
    5. Fix with ice cold 4% PFA in MgCl2

    Standard fixation time ~3h in cold room + ~30 min at RT.

     
  • Bruno Vellutini 18:02 on 2013/05/05 Permalink
    Tags: Lineus viridis   

    Fixation of 26d Lineus 


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    I had an egg mass with many worms. I tried relaxing for 20 min and they were not contracted, but also not fully extended. I then did a 2min 2% cystein wash followed by a FSW wash to remove the cystein. Worms started to move again, so I re-added MgCl2 and waited 5 min. After adding the fixative they contracted and became small balls :/

     
  • Bruno Vellutini 13:55 on 2013/05/02 Permalink
    Tags: Lineus viridis   


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    Cystein treatment for 12/04/13 (20d) egg mass. Most embryos were still inside the larval epidermis…

    03/05/13

    Fixed, but some did not look so good.

     
  • Bruno Vellutini 13:29 on 2013/05/01 Permalink
    Tags: , Lineus viridis   

    Lineus viridis in situ background 


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    First in situs in Lineus viridis showed an epithelial background of what it looks like mucus cells:

    Epidermis_insitu

    To avoid this I’m fixing with a cystein treatment pre-fixation in juvenile stages (details later on). However, to be sure that the background cells are the mucus cells and that cystein will work I had a look at some basic descriptions. Below it is a DIC image of the epidermis with some gland cells.

    Epidermis

    Comparing this image with the in situ pictures it looks like the background cells are below the epidermis (dermis) and that these droplets in the epidermis are serous cells of protein content. A diagram from (10.1093/icb/25.1.37) seems to confirm this for heteronemerteans:

    Norenburg1985_integument

    Bibliography

     
  • Bruno Vellutini 19:00 on 2013/04/30 Permalink
    Tags: Lineus viridis   


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    Cystein treatment for 12/04/13 (18d) and 24/04/13 (6d) egg masses. When opening the egg mass for 18d many juveniles poped-out  and thus received the cystein treatment directly.

    01/05/13

    Many juveniles (18d) burst because of the cystein treatment. I will need to fix this stage again. Otherwise, the earlier 6d embryos look fine.

     
  • Bruno Vellutini 15:54 on 2013/04/28 Permalink
    Tags: Lineus viridis   


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    Cystein treatment for 12/04/13 (16d).

    29/04/13

    Dissected and fixed.

     
  • Bruno Vellutini 19:32 on 2013/04/24 Permalink
    Tags: Lineus viridis   


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    Cystein treatment for 10/04/13 (14d).

    Also fixed a bunch of Meara juveniles for confocal (stored in my confocal fridge box) and in situs.

    26/04/13

    Fixed 14d.

     
  • Bruno Vellutini 18:11 on 2013/04/23 Permalink
    Tags: Lineus viridis   


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    Cystein treatment for 13/04/13 (10d) and 11/04/13 (12d).

    24/04/13

    Dissected and fixed both samples. 10d embryos were not looking so good, many seem to be loosing cells. 12d sample was great.

     
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