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  • Bruno Vellutini 18:45 on 2015/03/30 Permalink
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    Membranipora wnt in situ 


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    First new try with bryozoan in situ. Separate wells for early (until extended gastrula) and late stages (early cyphonautes and later cyphonautes).

    early stages 1x nk2.1 1x wnt1 1x wnt5 1x wnt7 1x wnt8
    late stages 0.1x nk2.1 0.1x wnt1 0.1x wnt5 0.1x wnt7 0.1x wnt8
    early stages 1x dlx 1x gbx 1x pax258 1x nanos 1x vasa
    late stages 0.1x dlx 0.1x gbx 0.1x pax258 0.1x nanos 0.1x vasa

    After protk step (10 min) embryos were suspended in glycine (added 800 µL) and did not sink. I added 600 µL of glycine for the second wash. The whole glycine steps lasted for 15 min. The remaining washes were a bit longer than 5 min, but I could control it better.

    I noticed that the embryonic membrane dissolves with protk.

    Let overnight in pre hybe.

    31/03/2015

    Diluted and added probes to in situ (first 0.1x then 1x). Left oven set to 63 °C (62 in the electronic thermometer and 63 in the mercury thermometer).

    02/04/2015

    First hybe wash I removed 1x probes first, filled with warm hybe wash and then removed 0.1x probes and added warm hybe.

    • 1x 10min hybe wash
    • 1x 50min hybe wash
    • 1x 30min 75% hybe
    • 1x 30min 50% hybe
    • 1x 30min 25% hybe
    • 1x 30min 2x SSC
    • 3x20min 0.2x SSC
    • 1x 10min 75% 0.2x SSC
    • 1x 10min 50% 0.2x SSC
    • 1x 10min 25% 0.2x SSC
    • 1x 10min PTw.
    • 4×10-15min washes in PBT (total 45min)
    • 1h in 1x blocking buffer
    • Overnight at 4°C with anti-dig-ap

    03/04/2015

    • 3x 5min PBT
    • 4x 15min PBT
    • 5x 30min PTw

    Developed in situ. A significant amount of pinkish background came up in my probes. wnt5 was fast, also dlx. Others had different levels of background in addition to the real signal, except Chema’s probe for NK2.1. Thus, this background is likely due to probe design or probe synthesis. Here are the lengths of the clones used to synthesize the probes:

    gene nk2.1 wnt1 wnt5 wnt7 wnt8 dlx gbx pax2/5/8 nanos vasa
    bp 1113 1038 1073 869 886 823 888 902 868 812

    Length of the probe can be the issue but it does not quite explain the rapid background coming up. Longer probes should result in a better signal to noise ratio because the signal comes up faster and not because the background comes up slower. It can also be something related to the probe synthesis process. But what? I usually get high yields and good peak ratios. Check the numbers to see!

    Ah, one obvious thing is the probe concentration. NK2.1 probe is probably more diluted than the others since it is its second or third use. Thus, the next in situ should be lower concentration or higher temperature.

    04/04/15

    Continued developing wnt7 and gbx and stopped them after 3h. Ethanol washes with 20min in 100% and let in PTw overnight at 4° C because glycerol was not ready.

    05/04/15

    Putting them in 70% glycerol with DAPI. Will put apart some of the NK2.1 embryos to do a MAPK immuno staining.

     
  • Bruno Vellutini 18:48 on 2014/11/22 Permalink
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    Terebratalia segmentation in situ 


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    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
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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 18:33 on 2014/11/07 Permalink
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    Novocrania cloning 


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    Set PCR for gbx, pax6, pax3/7, nk1, notch, delta.

    08/11/14

    Ran gel and set ligation.

    2014-11-08 15.29.44

     
  • Bruno Vellutini 17:31 on 2014/09/09 Permalink
    Tags: , , nk, , ,   

    Round of Terebratalia cloning 


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    I have ordered additional primers to clone more segmentation genes. Setting a PCR with 25 µM for primer concentration and 65 °C for annealing temperature.

    Ttra lmx1, nk1a, nk1b, odd

    10/09/14

    2014-09-10 12.10.25

    So, odd failed.

    Set ligation at 4 °C to go overnight (have to write…)

     
  • Bruno Vellutini 15:54 on 2012/09/14 Permalink
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    In situ of Terebratalia wnt genes 


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    Starting in situ for wnt genes and segmentation genes:

    wnt1 wnt9 gli
    wnt5 wnt10 smo
    wnt6 wnt16 nk6

    All genes took 2 days to developed, except for wnt9, wnt10 and smo. I let them all in AP -MgCl2 for the weekend and resumed developing on Monday morning. I exchanged the 3 wells for new AP substrate and let overnight at 4 °C.

     
  • Bruno Vellutini 16:54 on 2012/08/02 Permalink
    Tags: apical organ, , , , , nk, orthopedia,   

    RACE PCR for Pc gmcl, Mm nk2.1, otp, bruno 


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    Setting the first round of RACE PCR for:

    Pc gmcl1 F1/F2, Pc gmcl2 R1/R2, Mm nk2.1 F1/F2, Mm otp R1/R2, Mm celf2 F1/F2.

    Letting overnight.

    03/08/12

    Extraction and ligation.

    04/08/12

    Transformation and plating.

    06/08/12

    Colony PCR

    13/08/12

    Sequencing reaction for Pc gmcl genes.

    BV177 GMCL1 T7
    BV178 GMCL1 T7
    BV179 GMCL1 T7
    BV180 GMCL1 T7
    BV181 GMCL1 T7
    BV182 GMCL2 T7
    BV183 GMCL2 T7

     
  • 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.

     
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