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

    Terebratalia additional in situ of treated embryos 

    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: , , ,   

    Immuno staining for azak and dmh1 treated Terebratalia 

    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 18:52 on 2015/02/13 Permalink
    Tags: , , , serotonin, , synapsin, , tyrosinated tubulin   

    Brachiopod mesoderm immunostaining 

    Mesoderm and maybe some neurons and muscles. Engrailed antibody second try
    Ttra mixed stages: DAPI / PHALL / Tyros. Tub. 1:500 a594 (mouse) / Synapsin II 1:500 a647 (rabbit) Ttra mixed stages DAPI / PHALL / Serotonin 1:500 a647 (rabbit) / Engrailed 4D9 1:25 a594 (mouse)
    Nano mixed stages: DAPI / PHALL / Tyros. Tub. 1:500 a594 (mouse) / Synapsin II 1:500 a647 (rabbit) Nano mixed stages DAPI / PHALL / Serotonin 1:500 a647 (rabbit) / Engrailed 4D9 1:25 a594 (mouse)
    • 2h permeabilizing in 0.2 % PTx
    • 2h 0.2 % PTx + BSA.
    • 1h 0.2 % PTx + 5 % NGS.
    • Incubated with primary antibodies at 4°C in 0.2% PTx+NGS.

    14/02/15

    • Washed PTx+BSA 3x 5 min.
    • Washed PTx+BSA 4x 30 min.
    • Incubated 1h in PTx+NGS.
    • Added secondaries (alexa 594 mouse and alexa 647 rabbit) in a concentration of 1:200.

    15/02/15

    • Washed PTx+BSA 3x 5min.
    • Washed PBT 5x 10 min.
    • Stained with BODIPY FL and DAPI (1:500) for 2h.
    • Washed out staining with 1x PBS.
    • Overnight at 4 °C.

    16/02/15

    Mounting with Murray Clear went well.

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

    Azakenpaullone treated Terebratalia 

    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.

     
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