Tagged: Terebratalia transversa Toggle Comment Threads | Keyboard Shortcuts

  • Bruno Vellutini 11:13 on 2015/05/12 Permalink
    Tags: , , , Terebratalia transversa,   

    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 18:01 on 2015/02/27 Permalink
    Tags: , , , , , , Terebratalia transversa,   

    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: , , , Terebratalia transversa   

    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, Terebratalia transversa, 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: , , , , Terebratalia transversa   

    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.

     
  • Bruno Vellutini 12:49 on 2015/01/30 Permalink
    Tags: , , , , , , , Terebratalia transversa,   

    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: , , , , , , Terebratalia transversa,   

    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 10:54 on 2014/11/27 Permalink
    Tags: , , , , , , , , pou, , Terebratalia transversa   

    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 18:48 on 2014/11/22 Permalink
    Tags: , , , , , , , , Terebratalia transversa,   

    Terebratalia segmentation in situ 

    In order to have more temporal resolution of engrailed I am adding one well per stage. In addition some segmentation related genes.

    en cleavage+blastula en radial gastrula en asym gastrula en bilateral gastrula en trilobed+early+late larva
    pax6 nk1a nk1b lmx1
     lfng runx hh

     25/11/14

    Washed probe out. Added antiDIG-AP antibody at 1700 and incubated overnight at 4 °C.

    26/11/14

    Washed antibody and started developing. Pax6 came up quite fast. I exchanged the solution after 1h and stopped after 2h. The remaining are slowly coming up, except asym and bilateral of engrailed. I used one tube of 0.8 ng/µL of probe and it must have been even lower concentration because the other 3 engrailed wells are coming up ok (same probe batch, but from another tube). Another issue is that there is not early gastrula like I wanted, they seem to be already have the differentiated lateral patches.

    Lunatic fringe and runx are not showing up. I exchanged AP from all wells except engrailed after 3h and then all wells before leaving at 4 °C overnight.

    27/11/14

    Same as yesterday with NK1s and lmx coming up nicely. Exchanged AP at 10:30.

    28/11/14

    Kept developing for 7h and stopped all wells. Nothing came up for lfng, runx or hedgehog.

    30/11/14

    Ethanol washes for all. Added 70% glycerol with 1:10000 sytox green in hedgehog well. I want to see if embryos turn red… If not I’ll check if it is better than DAPI for these regular in situs.

     
  • Bruno Vellutini 19:31 on 2014/11/18 Permalink
    Tags: , , , , , , , , Terebratalia transversa   

    Probe synthesis of Novocrania Hedgehog pathway 

    Selected minipreps for probe synthesis of Novocrania Hedgehog pathway and remaining Terebratalia genes. From this post.

    BV326 Terebratalia transversa Lmx1 T7 ok + SP6
    BV328 Terebratalia transversa NK1a T7 ok + SP6
    BV330 Terebratalia transversa NK1b T7 ok + SP6
    BV332 Novocrania anomala Patched1 T7 ok + SP6
    BV336 Novocrania anomala Smoothened1 T7 ok + SP6
    BV338 Novocrania anomala Smoothened2 T7 ok + SP6
    BV340 Novocrania anomala Gli T7 ok + SP6
    BV343 Novocrania anomala Pax6 T7 ok + SP6
    BV345 Novocrania anomala Pax3/7 T7 ok T7
    BV346 Novocrania anomala NK1 T7 ok + SP6
    TTR Terebratalia Pax6 T7 DIG
    TTR Terebratalia Pax6 T7 DNP

    Set probe PCR to run overnight.

    19/11/14

    Ran gel and painfully extracted the PCR…

    2014-11-19 19.09.46

    20/11/14

    Started probe synthesis for the genes above at 9:30. Finished at 16:30 and precipitated overnight.

    21/11/14

    gene ng/µL goal concentration volume total volume volume of hybe buffer
    Ttra lmx1 906 50 24 426 402
    Ttra nk1a 974 50 24 458 434
    Ttra nk1b 1098 50 24 516 492
    Nano ptc1 1075 50 24 505 482
    Nano smo1 2041 50 24 959 936
    Nano smo2 1830 50 24 860 836
    Nano gli 488 50 24 229 206
    Nano pax6 1350 50 24 634 611
    Nano pax37 2224 50 24 1045 1022
    Nano nk1 1050 50 24 494 470
    Ttra pax6 dig 1076 50 24 506 482
    Ttra pax6 dnp 50 50 24 23 0

     

     
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