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  • Bruno Vellutini 12:49 on 2015/01/30 Permalink
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    In situ Novocrania and Terebratalia 


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    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 13:36 on 2015/01/19 Permalink
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    Cloning more segmentation genes 


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    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
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    Novocrania and double in situs of Terebratalia 


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    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 19:31 on 2014/11/18 Permalink
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    Probe synthesis of Novocrania Hedgehog pathway 


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    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

     

     
  • Bruno Vellutini 18:49 on 2014/11/11 Permalink
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    Transformation to sequencing of Novocrania 


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    I selected the 8 most important Novocrania genes from the first and second batched of cloning: ptc1, ptc2, smo1, smo2, gli, pax6, pax3/7, nk1

    Transformed and plated.

    12/11/14

    Set colony PCR:

    2014-11-12 15.43.52

    13/11/14

    BV332 Novocrania anomala Patched1 T7 ok + SP6
    BV333 Novocrania anomala Patched1 T7 ok + SP6
    BV334 Novocrania anomala Patched2 T7 ?
    BV335 Novocrania anomala Patched2 T7 ?
    BV336 Novocrania anomala Smoothened1 T7 ok + SP6
    BV337 Novocrania anomala Smoothened1 T7 ok + SP6
    BV338 Novocrania anomala Smoothened2 T7 ok + SP6
    BV339 Novocrania anomala Smoothened2 T7 ok + SP6
    BV340 Novocrania anomala Gli T7 ok + SP6
    BV341 Novocrania anomala Gli T7 ok T7
    BV342 Novocrania anomala Pax6 T7 ok T7
    BV343 Novocrania anomala Pax6 T7 ok + SP6
    BV344 Novocrania anomala Pax3/7 T7 ok T7
    BV345 Novocrania anomala Pax3/7 T7 ok T7
    BV346 Novocrania anomala NK1 T7 ok + SP6
    BV347 Novocrania anomala NK1 T7 ok + SP6

     

     
  • Bruno Vellutini 16:15 on 2014/11/10 Permalink
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    Cloning Hedgehog components of Novocrania 


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    PCR with diluted primers for 40 cycles instead of 34 for: ptc1, ptc2, smo1, smo2, gli

    2014-11-10 22.32.27

    Had two bands for some. Set a ligation overnight for all at 4 °C.

     
  • Bruno Vellutini 19:19 on 2012/11/13 Permalink
    Tags: , , patched,   


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    Took pictures of hedgehog and patched Terebratalia in situs. I think I covered the missing positions and stages.

     
  • Bruno Vellutini 15:22 on 2012/05/25 Permalink
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    In situ segmentation genes Terebratalia 


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    Starting an in situ for segmentation genes in Terebratalia and repeating the fluorescent in situ for Vasa and Nanos, trying to reduce the background. The plate looks like this:

    netrin patched hedgehog vasa
    smo2 nk2.2 nk5 nanos

    For vasa and nanos I will use a lower concentration of the antibody, 1:500 (last time it was 1:100) — effort to reduce background. For nanos I will also double the amount of probe to get a stronger signal.

    Samples put in prehybe at 17h:00.

    26/05/12

    Added regular probes at 1 ng/µL, except for nanos which was 2 ng/µL (trying to increase the signal/noise ratio for fluorescent in situ).

    28/05/12

    Washes. Added regular Anti-Dig/AP to segmentation genes (1:5000) and Anti-Dig/POD (1:250) to vasa and nanos (in an attempt to lower the background noise — used 1:100 previously).

    29/05/12

    Regular washes with PBT and PTw.

    30/05/12

    Started developing segmentation genes of Terebratalia at 11:30. For the fluorescent in situ I developed normally for 1h30 and then stopped the reaction with a detergent solution used by (10.1038/nature10838) to reduce the background; 3 washes of 20 min at 62 °C. The solution:

    [list of chemicals]

    I checked the slides in the fluorescent lamp and did not see any staining. It is also not so clear if there is less background. I guess I need to check in the confocal.

    The segmentation genes began to appear. Netrin is the strongest one and the pattern is interesting. I notice that the “ring” background might be covering the whole anterior of the pedicle lobe. Other genes are still weak to say something.

    Changed the AP substrate at 16:30 and let it developing at 4 °C.

    31/05/12

    Patterns are more clear today. Changed the substrate 3 times (10:15, 12:30, 16:00) and let it overnight at 4 °C. Crystal began to appear after lunch and are already covering the whole bottom of the well.

    01/06 – 04/06

    Stopped netrin, nk2.2, and nk5. Developed others until 04/06. Many crystals forming…

    Bibliography

     
  • Bruno Vellutini 12:03 on 2012/05/18 Permalink
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    Terebratalia in situ for germline genes (+Membranipora testing) 


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    First in situ using segmentation genes for Terebratalia transversa. The list is:

    Tt netrin BV110 SP6
    Tt patched BV115 SP6
    Tt hedgehog BV129 SP6
    Tt smoothened2 BV131 SP6
    Tt nk2.2 BV135 SP6
    Tt nk5 BV139 SP6
    Mm piwi1 BV94 SP6
    Mm gata123

    Put in prehybe at 19:45.

    19/05/12

    Probes for Terebratalia genes were no good, so changing to Vasa, Nanos and Piwi:

    Tt vasa
    Tt nanos
    Tt piwi
    Tt vasa (fluo)
    Tt nanos (fluo)
    Tt piwi (fluo)
    Mm piwi1
    Mm gata

    Put the probes and let hybridizing at hybe temp.

    21/05/12

    First day of washes. Added Anti-Dig/POD (1:100) to the 3 wells of vasa, nanos, and piwi and the regular Anti-Dig/AP for the remaining; 19h and let overnight at 4° C.

    22/05/12

    Washing and started developing the regular in situ in 5 wells overnight at 4 °C.

    23/05/12

    Changes in AP Substrate: 10:30, 12:00, 14:30

    AP is becoming purpleish quite rapidly; patterns are developing accordingly fast and as expected for Terebratalia genes. Membranipora Piwi1 shows no sign of staining (although it seems to be a bit pinkish) and GATA123 is appearing, although it is not easy to identify the pattern (or if it is similar to Andi’s in situ).

    At 11:00 I started washing and then developing the fluorescent in situ. Checking under the 4D room scope the embryos show staining, but it is hard to say if the in situ worked, yet. I need to look mount them and look in the scope.

    24/05/12

    Mounted fluorescent in situ samples after staining with DAPI (1:1000) and checked in the scope. Vasa is the strongest, but still a bit weak. So next time it is better to use more probe. Nanos is there, but barely visible and Piwi is difficult to say, since the expression is widespread. Made images in the confocal and the signal seems to be there, but there is a lot of background.

    Stopped Vasa regular in situ at 10:00 and changed the substrate for the others. Changed again at 13:30. Yellow crystals were present in the Membranipora wells. Changed at 16:30 and left overnight at 4 °C.

    25/05/12

    Stopped all wells and stored in 70% glycerol.

     
  • Bruno Vellutini 17:48 on 2012/05/15 Permalink
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    Probe synthesis for Terebratalia segmentation 


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    Beginning probe synthesis for:

    Tt netrin BV110 SP6
    Tt patched BV115 SP6
    Tt hedgehog BV129 SP6
    Tt smoothened2 BV131 SP6
    Tt nk2.2 BV135 SP6
    Tt nk5 BV139 SP6
    Mm ago2 BV25 SP6
    Mm mago nashi BV27 T7
    Mm germ cell less BV30 T7

    Run the PCR and letting overnight to extract tomorrow.

    16/05/12

    Ran gel and extracted the bands. Looking ok:

    18/05/12

    Started probe synthesis reaction at 10:40. Precipitated around 16h.

    19/05/12

    Terebratalia probes had no pellet (but in fact, I don’t know if I centrifuged it). Membranipora using SP6 had a small pellet and the two T7 ones had a huge pellet. Specs are:

    Gene ng/µL
    Mm Ago2 293.92
    Mm Magoh 3806.73
    Mm GMCL 3147.70

    Diluted to 50 ng/µL and stored at -20 °C.

    21/05/12

    Re-doing the transcription reaction for Terebratalia segmentation genes. Started at 11h, but only put at 37°C at 13h (my bad…). Precipitated at 17h30 using only 5 µL of Lithium Chloride. Could not see any pellet…

    22/05/12

    After centrifuging a thin pellet appeared in the bottom of the tubes. I used Low yield, but enough for making probes:

    Gene ng/µL
    Tt netrin 120.56
    Tt patched 174.64
    Tt hedgehog 120.93
    Tt smo2 107.95
    Tt nk2.2 211.83
    Tt nk5 221.16

    Diluted to 50 ng/µL and stored in the freezer.

     
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