Tagged: Novocrania anomala Toggle Comment Threads | Keyboard Shortcuts

  • 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.

    13/05/15

    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.

    14/05/15

    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.

    12/05/15

    Set transcription reaction and precipitation.

    13/05/15

    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.

    09/05/15

    Added probes just after synthesis.

    11/05/15

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

    12/05/15

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

    13/05/15

    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

    07/05/15

    2015-05-07 11.35.14

    It worked. Extracted, ligated, transformed and plated.

    08/05/15

    Colony PCR and 2 volonies were fine:

    2015-05-08 17.18.24

    Put these to grow under the IDs:

    T7
    Na wnt1 long BV386
    Na wnt1 long BV387

    09/05/15

    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.

    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 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.

    31/01/2015

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

    02/02/2015

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

    03/02/2015

    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).

    04/02/2015

    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.

    05/02/2015

    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.

    28/01/2015

    Ran gel and extracted.

    2015-01-30 11.05.52

    29/01/2015

    Set probe synthesis and precipitated.

    30/01/2015

    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

    T7
    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)

     28/11/14

    Added probes at 1 ng/µL concentration.

    30/11/14

    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.

    01/12/14

    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).

    02/12/14

    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.

    26/11/14

    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

    28/11/14

    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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