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  • Bruno Vellutini 11:13 on 2015/05/12 Permalink
    Tags: , Novocrania anomala, , ,   

    Novocrania wnt1 in situ with longer probe 

    Na wnt1 Tt runx

    Did everything as usual and embryos looked normal.


    Embryos quite damaged, including Terebratalia. Possible that pipette is not well calibrated?

    Restarted in situ today with extreme gentleness. Added 5 µL protk in 10 mL (instead of 2.5 in 5 µL) using the P20 pipette. Fosfatase wash with 83 °C and added hybe buffer around 1800.


    Add probe.

  • Bruno Vellutini 22:57 on 2015/05/11 Permalink
    Tags: , Novocrania anomala, , ,   

    Long probe for Novocrania wnt1 

    Set probe PCR for:

    Na wnt1 BV387: T7 80 ng/µL
    Mm pl10 BV79: SP6 702 ng/µL

    and extracted.


    Set transcription reaction and precipitation.


    Finished probes (see values above) and made 1x dilution for wnt1.

  • Bruno Vellutini 18:24 on 2015/05/08 Permalink
    Tags: , , , , , , , Novocrania anomala, , , ,   

    Membranipora in situ and Novocrania wnt1 

    Starting in situ in Membranipora with dorsoventral genes and additional genes that we backgroundish.

    wnt1 dlx bmp2/4 chordin fgf
    nanos vasa piwi1 piwi2 pl10
    Na wnt1

    Standard in situ protocol with 10 min protk. Washes after glycine were slightly shorter and fixing went for 1:30h. Used 83°C for fosfatase step and prehybe at 67°C.


    Added probes just after synthesis.


    Novocrania were dissolved. The rest were fine. Continued with hybe washes and added anti-dig-ap.


    Antibody washes. Plus developing. dlx and piwi1 were stopped after a couple of hours. The remaining were developed overnight at 4 °C.


    Exchanged the AP and stopped the reaction around 1200. Did ethanol washes and left in 70% glycerol + dapi overnight at room temp.

  • Bruno Vellutini 20:07 on 2015/05/06 Permalink
    Tags: Novocrania anomala, , ,   

    Novocrania wnt1 one more time 

    Set PCR with 60°C annealing, 1:45 elongation, 40 cycles and primers at 25mM.

    wnt1 1:10 cDNA wnt1 1:20 cDNA
    pax6 1:10 cDNA pax6 1:20 cDNA


    2015-05-07 11.35.14

    It worked. Extracted, ligated, transformed and plated.


    Colony PCR and 2 volonies were fine:

    2015-05-08 17.18.24

    Put these to grow under the IDs:

    Na wnt1 long BV386
    Na wnt1 long BV387


    Extract miniprep and set sequencing reaction.

  • Bruno Vellutini 18:52 on 2015/02/13 Permalink
    Tags: , , Novocrania anomala, 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.


    • 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.


    • 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.


    Mounting with Murray Clear went well.

  • Bruno Vellutini 12:49 on 2015/01/30 Permalink
    Tags: , , , , Novocrania anomala, , , ,   

    In situ Novocrania and Terebratalia 

    In situ using Nano longer probes to try to get a better signal to ratio. Also fgf8 probe, important for the brain boundary. I also need better hedgehog pictures, so I’m repeating the in situ with a new probe in Ttra and also trying to get a good early gastrula, bilateral gastrula e late larvae of engrailed.

    Nano wnt5 Nano smo2 Nano fgf8 Nano ptc1
    Ttra hh Ttra en late blastula / early gastrula Ttra en bilateral gastrula / late larva

    10 min ProtK for all. Incubated at 65 °C at 2000.


    Diluted probes to 1 ng/µL and added at 0800.


    First day of washes. Added anti-dig-ap at 1600.


    Washed off antibody with 3x 5min followed by 4x 15 min and 5x 30 min. Developed with nbt/bcip at 1600. All wells showed good signal/noise ratio. The worst is wnt5, but it is still better than the other in situ. I exchanged the AP for all wells at 1930 and stopped all except hedgehog and en bilateral (exchanged AP).


    Exchanged the AP for the two Terebratalia wells, I’ll let them develop longer.

    Stopped at 1500 and did ethanol washes. Let overnight in 70% glycerol.


    Mounting and pictures.

  • Bruno Vellutini 18:15 on 2015/01/27 Permalink
    Tags: , , , Novocrania anomala, , , ,   

    Probe synthesis of Terebratalia and Novocrania 

    Probe synthesis of some longer fragments for Novocrania in situ wnt5, smo, fgf8 and standard Terebratalia clones hh and en.

    SP6 ng/µL
    Na wnt5 BV352  1146.5
    Na smo2 BV354  906.7
    Na fgf8 BV355  964.2
    Tt hh BV129  1510.6
    Tt en TTR54  573.1

    Set probe PCR. Extracted.


    Ran gel and extracted.

    2015-01-30 11.05.52


    Set probe synthesis and precipitated.


    Finished probes.

  • Bruno Vellutini 13:36 on 2015/01/19 Permalink
    Tags: , , Novocrania anomala, , , ,   

    Cloning more segmentation genes 

    I selected alternate primers for getting longer fragments and improving the quality of Novocrania in situs.

    Nano ptc2, wnt1, smo1, smo2, fgf8, Mmem wnt4

    2015-01-20 11.02.32

    wnt1 did not work again, bad ptc2 again, smo1 and smo2 weakly worked, fgf8 worked, Mmem wnt4 not… Re-run Nano ptc2 with different primers:

    2015-01-20 17.51.33

    Combination of primers 3F and 2R (395 bp fragment) was the only one that worked. Transformed:

    2015-01-23 17.16.01

    Na wnt5 BV352
    Na wnt5 BV353
    Na smo2 BV354
    Na fgf8 BV355
    Na fgf8 BV356
    Na ptc2 BV357
    Na ptc2 BV358


  • Bruno Vellutini 10:54 on 2014/11/27 Permalink
    Tags: , , , , , Novocrania anomala, , , pou, ,   

    Novocrania and double in situs of Terebratalia 

    Nano ptc1 Nano smo1 Nano smo2 Nano gli
    Nano pax6 Nano delta Nano notch
    Ttra pax6 (DNP/cy5) + pax258 (DIG) Ttra en (DNP) + pax6 (DIG) Ttra wnt1 (DNP) + delta (DIG) Ttra wnt1 (DNP) + pou4 (DIG)


    Added probes at 1 ng/µL concentration.


    Washes, incubated with the blocking for 1h and at 4 °C overnight with the respective antibodies. Anti-DIG-AP 1:5000 for all Novocrania wells and Anti-DIG-POD 1:250 for all Terebratalia wells.


    Washed antibody from all samples with:

    • 5x 15 min PBT and 5x 30 min PTw.
    • 3x 5min washes of AP minus MgCl2 for regular in situ and TNT buffer for fluorescent in situs.

    Developed fluorescent wells with TSA kit using cy3 fluorochrome for 2 hours. Sropped the reaction with 5x 5 min detergent solution at 60°C followed by PTw washes. POD inactivation with 0.1% H2O2 for 45 min, PTw washes, and finally formamide based POD inactivation buffer for 15 min at 60 °C. Signal for pax6 and delta were very strong, pou4 was strong and pax258 was medium.

    I started developing Novocrania with NBT/BCIP. Pax6 was the first to come, strongly. The others were slower, but gli had a good signal/noise ratio. Others acquired background after the first AP change after 2 hours. I stopped all, except delta and smo2 (which had less background).


    Today they were quite dark :/

    Went through ethanol washes and antibody washes for fluorescent. Added 70% glycerol in PTw with 1:10000 sytox green.

  • Bruno Vellutini 15:31 on 2014/11/25 Permalink
    Tags: , Novocrania anomala, ,   

    Cloning Novocrania patched and longer clones of wnt1, wnt5 

    I made new primers for ptc2 and longer clones for wnt1 and wnt5. Set PCR overnight.


    Ran gel and cut a band, but wnt1 and ptc2 did not work… OF COURSE! because fragment lengths are the following: wnt1=1239 bp, wnt5=1097 bp, ptc2=1258 bp! And the elongation time of the PCR was set to X.

    2014-11-26 11.16.21


    Re-tried the same PCR with a longer elongation time. The result was the same. Chema suggested running nested primers to see if something appears.

    2014-11-28 17.18.15

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