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  • Bruno Vellutini 18:01 on 2015/02/27 Permalink
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    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 18:01 on 2015/02/26 Permalink
    Tags: , , inhibitors,   

    Immuno staining for azak and dmh1 treated Terebratalia 


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    Primary: Tyros. Tub. 1:500 (a594 mouse) / Synapsin II 1:500 (a647 rabbit).

    Secondary: Alexa 594 (mouse) / Alexa 647 (rabbit) in a concentration of 1:200.

    Staining: DAPI 1:500/ PHALL for 2h.

    Mid blastula

    1µM 10µM
    control  dmh1 treated

    Early gastrula

    1µM 10µM
    control  dhm1 control

    Mid blastula to larva

    1µM 10µM
    control
    • Washed and cleaned embryos many times ~1h.
    • 1600 BSA 2x
    • 1730 NGS
    • 1830 Incubate

    27/02/2015

    • 3x 5 min 0.2% PTx+BSA
    • 4x 30 min 0.2% PTx+BSA
    • 2h 0.2% PTx+NGS
    • Added A594 (mouse) and A647 (rabbit) 1:200.

    28/02/2015

    • 3x 5 min 0.2% PTx+BSA
    • 4x 30 min 0.2% PTx+BSA
    • 2h staining in BODIPY FL and DAPI
    • 4x 5min 1x PBS

    01/03/2015

    Mounted in Murray and confocal-ed.

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

    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 13:06 on 2014/09/23 Permalink
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    4D with U0126 Membranipora 


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    15 °C, 15 µM

    BCV_Mm_2014_09_23 (old Mmem1_23_09_2014)

     
  • Bruno Vellutini 15:38 on 2014/09/20 Permalink
    Tags: inhibitors, , ,   

    Membranipora Endo-IWR-1 


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    Trying the Endo-IWR-1 to inhibith the Wnt Pathway. Setting 2 plates with 10, 20, and 40 µM.

    For 24h

    DMSO 10 µM
    20 µM 20 µM

    For 48h

    DMSO 10 µM
     40 µM 40 µM

    I miss calculated the well and put 20 µM on the last well of the first plate. So I set 40 µM for the third and fourth wells of the second plate.

    21/09/14

    I checked the embryos and it is hard to see if there is any phenotype. I’m letting it go another day to fix.

    22/09/14

    Fixed at 48h.

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

    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 13:00 on 2014/09/20 Permalink
    Tags: , inhibitors, , ,   

    4D with U0126 Membranipora 


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    BCV_Mm_2014_09_20 (old Mmem1_20_09_2014)

    10 µM, 15 °C

     
  • Bruno Vellutini 15:34 on 2014/09/19 Permalink
    Tags: inhibitors, , ,   

    Membranipora U0126 inhibitor time windows 


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    I set the U0126 inhibitor 10 µM with Membranipora at 15 °C. I am washing every 2 hours after 1h30 hours after activation as shown below (2x, for 24h and 48h fixation).

    • Activated: 1100
    • Inhibited: 1230
    • 1st wash: 1430
    • 2nd wash: 1630
    • 3rd wash: 1830
    DMSO 3.5 hpa
    5.5 hpa 7.5 hpa

     20/09/14

    Fixed 24h.

    21/09/14

    Fix 48h.

     
  • Bruno Vellutini 17:07 on 2014/09/18 Permalink
    Tags: , inhibitors, , ,   

    Membranipora 4D U0126 10 µM 


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    Started a recording BCV_Mm_2014_09_18 (old Mmem1_18_09_2014) with Membranipora in 10 µM U0126 MEK inhibitor at 15°C. Embryo is looking quite good and stable.

    19/09/14

    Embryo is still lokking good and healthy (?). Letting it on… Stopped and saved the embryo. It moved out of the field of view around 1700.

     
  • Bruno Vellutini 16:34 on 2014/09/17 Permalink
    Tags: inhibitors, , , ,   

    Membranipora Wnt inhibitors 


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    Second round of testing inhibitors and activators of the Wnt pathway in Membranipora. Firt one was last year and they all showed a disrupted phenotype. I am repeating some treatments. Started with 2h post activation. 15 °C.

    Azakanpaullone (x2)

    DMSO 1 µM Azakanpaullone
    2.5 µM Azakanpaullone 5 µM Azakanpaullone

    IWR-1

    DMSO 10 µM IWR
    20 µM IWR 40 µM IWR

    PNU74654

    DMSO 1 µM PNU
    10 µM PNU 25 µM PNU

     18/09/14

    Fixed one plate of Azakanpaullone at 24h. Leaving the others to be fixed at 48h.

    Azak: seems to give a concentration dependent phenotype, but it is quite hard to identify what is wrong.

    IWR & PNU: Although higher concentrations seem to have disturbed the development somehow, it is not clear what is wrong. Maybe there is nothing wrong. I’m leaving them for 48h, maybe a phenotype becomes clearer.

    19/09/14

    Azak: 1 µM has a mild phenotype with a reduced early larva. 2.5 µM shows ciliated swimming balls or some partly differentiated reduced larvae. I am not sure if it is a ventralized phenotype, but it seems that the apical organ is missing. 5 µM balls of cells, not swimming, maybe already dead.

    IWR & PNU: Not sure if there is any phenotype…

    Fixed all for 1 hour at room temperature.

     
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