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  • Bruno Vellutini 18:01 on 2015/02/27 Permalink
    Tags: azakenpaullone, , , , , , ,   

    Terebratalia additional in situ of treated embryos 


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    wnt1 azak control (0.1 ng/µL)

    mid blastula to larva

    engrailed azak control (0.1 ng/µL)

    mid blastula to larva

    pax6 azak control (0.1 ng/µL)

    mid blastula to larva

    wnt1  azak 10µM

    mid blastula

    pax6 azak 1µM

    mid blastula

    engrailed azak control

    early blastula

    engrailed azak control

    mid blastula

    wnt1 dmh1 engrailed dmh1 pax6 dmh1
    wnt1 dmh1 control engrailed dmh1 control pax6 dmh1 control

    Started in situ as normal. Except dmh1 wells stayed in protk for 15 min and not 10… last 5 minutes in the nutator, so they maybe are a bit digested.

    28/02/15

    Added probes around 1700.

    02/03/15

    Hybe washes. Dorsomorphin embryos started to dissolve in the SSC and I switched to gentle mode. By the end of the day there was not much left, but a few embryos survived. Added anti-dig-ap and left overnight.

    03/03/15

    Antibody washes went fine and started developing at 1500.

    Only relatively normal well developing was dorsomorphin pax6 and some dmh1 controls, but it seems that the embryos have been overly digested. Epidermis looks bad.

    Switched the AP once and let overnight.

    04/03/15

    Some signal is appearing in some controls, but overall it still looks bad. Exchanged AP at 1000, 1530 and X.

     
  • Bruno Vellutini 20:09 on 2015/02/11 Permalink
    Tags: azakenpaullone, , , ,   

    Azakenpaullone treated Terebratalia 


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    Another Terebratalia in situ with Azakenpaullone treated embryos to check the localization of gene products. I want to see if the positioning of Pax6 and Pax2/5/8 has been affected by the displacement of the wnt pathway.

    Blastula Mid Blastula Mid blastula to larva Early Gastrula Early Larva
    1 µM 1 µM 1 µM 1 µM 1 µM
    10 µM 10 µM 10 µM 10 µM 10 µM
    C- C- C- C- C-

    Proceeded normally with new 85 °C to remove fosfatases and incubated at 67°C.

    12/02/15

    Added probes.

    14/02/15

    Hybe washes went normally according to the protocol. Added Anti-DIG/AP at 2000.

    15/02/15

    Washed antibody many times with PBT 5x 15 min and 3x 30 min in PTw and let overnight at 4 °C around 1300.

    16/02/15

    Started developing at 0930. Signal is quite weak in the first 5 hours.

     
  • Bruno Vellutini 15:34 on 2014/09/20 Permalink
    Tags: azakenpaullone, , ,   

    Membranipora Azakenpaullone for in situ 


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    Prepared large spawning of Membranipora for Azakenpaullone treatment at 1, 2.5 and 5 µM in 4 mL volume of sea water.

    DMSO 1 µM
    2.5 µM 5 µM

    To be fixed at 24 and 48h.

     
  • Bruno Vellutini 08:00 on 2014/04/05 Permalink
    Tags: azakenpaullone, , , , ,   

    Terebratalia Azakenpaullone treated in situ wnt1 and engrailed 


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    Starting in situ with azakenpaullone-treated Terebratalia embryos with wnt1.

    Blastula Mid Blastula Mid blastula to larva Early Gastrula Early Larva
    1 µM 1 µM 1 µM 1 µM 1 µM
    10 µM 10 µM 10 µM 10 µM 10 µM
    C- C- C- C- C-

    Prepared fresh PTw and went up to hybe step. Then split samples for wnt1 and engrailed (30 wells in total). Wnt1 probe will be fine, but engrailed will be much less concentrated, probably around 0.1 ng/µL. Unfortunately this is what I had in stock.

    06/04/14

    I fell face to the ground and could not go to the lab. Chema put my 2 plates in the freezer.

    07/04/14

    I put the plates back at 62 °C for 5h. I diluted wnt1 probes and added the usual 500 µL volume to the wells. For the engrailed probe I had to dilute it to 0.8 ng/µL and use only 250 µL of volume… but well.

    09/04/14

    Washing day. Everything went normally. I added the antibody around 17:30.

    10/04/14

    Washed 2 x 15 min and 1x 1h with PBT and then 1x 15 min. Finally 5x 30 min with PTw. Left the plates in the cold room.

    11/04/14

    Developing day. Ran for 5h and some signal is appearing; stronger in the treated embryos than in the controls. Exchanged the AP substrate at 17:30 and put in the cold room.

    12/04/14

    Most of the treated embryos have a nice signal. Controls are surprisingly faint, though. I developed through the whole day and kept in the cold room overnight. Probably stopping tomorrow some wells.

    13/04/14

    Stopped the reactions in the treated samples with 3 washes of AP without magnesium chloride. I’m still developing the controls.

    14/04/14

    Still developing controls. Stopped wnt1, but not engrailed.

    15/04/14

    Exchanged again the AP. It looks much better now, signal is stronger. I did not filter the AP this time to see if I get crystals.

    Developed for 8h and stopped the reaction on the last wells.

    16/04/14

    Ethanol washes upt to glycerol.

     
  • Bruno Vellutini 17:30 on 2013/09/24 Permalink
    Tags: , azakenpaullone, ,   

    4D Membranipora Azakenpaullone 5 µM 


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    Folder: BCV_Mm_2013_09_24

    Started a recording with Membranipora with 5 µM Azakenpaullone. Relatively vegetal view.

    Around 18h the embryo detached and was shaking every slice… impossible to track. Kept it overnight just in case.

    25/09/13

    Stopped, embryo was gastrulating.

     
  • Bruno Vellutini 18:34 on 2013/08/30 Permalink
    Tags: , azakenpaullone, ,   

    4D Membranipora Azakenpaullone 5 µM 


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    Folder: BCV_Mm_2013_08_30

    Another recording with Az. Kind of lateral view and optics are good.

    31/08/13

    Looking good. It became something.

    02/08/13

    Stopped the recording. Deleted from lineage station stack X3201 onwards, embryo was getting bad.

     
  • Bruno Vellutini 19:27 on 2013/08/29 Permalink
    Tags: , azakenpaullone, ,   

    4D Membranipora Azakenpaullone 5 µM at 15 °C 


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    Folder: BCV_Mm_2013_08_29

    Set a recording with azakenpaullone 5 µM. Embryo at 2 cell, looking ok.

    30/08/13

    Checked the history and during the second division 1 blastomere fails to divide. Subsequent divisions look kind of normal, but I presume this is not due to the inhibitor.

    Stopped the recording in the afternoon.

     

     
  • Bruno Vellutini 22:39 on 2013/08/16 Permalink
    Tags: azakenpaullone, , , ,   

    Trying other inhibitors with Membranipora 


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    I spawned and picked embryos (16/08/13 A: 17h30) for other inhibitors related to the Wnt pathway. Here is the setup put at 10 °C with inhibitors at 21h30 (4h after activation).

    2013-08-16 21.34.32

    DMSO 1:500 Azakenpaullone 1 µM
    Azakenpaullone 5 µM Azakenpaullone 10 µM
    DMSO 1:500 IWR-1 10 µM
    IWR-1 20 µM IWR-1 40 µM
    DMSO 1:500 PNU74654 1 µM
    PNU74654 10 µM PNU74654 25 µM

    18/08/13

    Fixed samples today and from the phenotypes I think I have the best concentrations for further experiments.

    • Azakenpaullone: 5 µM
    • IWR-1: 20 µM
    • PNU74654: 25 µM
     
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