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  • Bruno Vellutini 10:36 on 2013/12/16 Permalink
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    Propidium+Phallacidin and Murray Clear for Novocrania engrailed fluorescent in situ 


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    Since mounting with TDE was not so good (crystals and some weird noisy background) I am staining with propidium iodide and phallacidin and mounting with murray clear. This should give a clean embryo with the mesodermal signal visible enough for details. Fluorochrome for engrailed is Cy5.

    17/12/13

    Confocal.

     
  • Bruno Vellutini 13:17 on 2013/12/08 Permalink
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    Terebratalia and Novocrania in situ 


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    Began in situ 10 min protk (half-concentrated for Nano) with:

    Ttra wnt2 Ttra wnta  
    Nano wnt8 Nano wnt11 Nano pax2/5/8

    07/12/13

    Added probes at 13:00.

    09/12/13

    First day of washes, added anti-dig-ap around 18h.

    10/12/13

    Washing off antibody, starting with 2x 15min PBT and after 1h 3x 15min PBT and normal PTw washes.

    Started developing at 16h. Some signal started to appear at Wnt11 ~18h30.

    11/12/13

    Arrived 9h30 and the wells were purple! Did an extra wash with AP substrate and let at RT. Exchanged AP 4 times and stopped the reaction at 19h with 3x AP without MgCl2 and 5x PTw. Kept at 4 °C overnight.

    12/12/13

    Ethanol washes, then first wash with glycerol and let in nutator for 3h. Exchange glycerol for glycerol+dapi and let it overnight.

    13/12/13

    Mounted slides of Novocrania and took pictures.

     
  • Bruno Vellutini 11:02 on 2013/12/04 Permalink
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    Probe synthesis for Novocrania and Terebratalia 


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    Probe PCR with 6 samples:

    2013-12-04 18.40.07

    05/12/13

    Set transcription reaction for all except for MP68 at 10h30. Began precipitation with 5 µL of lithium.

    06/12/13

    ng/µL
    Ttra wnt2 1008
    Ttra wnta 801
    Nano wnt8 2000
    Nano wnt11 1353
    Nano pax258 860

     
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