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  • Bruno Vellutini 18:45 on 2015/03/30 Permalink
    Tags: , , , , , , pax, ,   

    Membranipora wnt in situ 

    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 18:01 on 2015/02/27 Permalink
    Tags: , , , , , pax, ,   

    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 20:09 on 2015/02/11 Permalink
    Tags: , , , pax,   

    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 10:54 on 2014/11/27 Permalink
    Tags: , , , , , , , pax, 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 18:48 on 2014/11/22 Permalink
    Tags: , , , , , , pax, , ,   

    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: , , , , , pax, , ,   

    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

     

     
  • Bruno Vellutini 18:49 on 2014/11/11 Permalink
    Tags: , , , pax,   

    Transformation to sequencing of Novocrania 

    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
    Tags: , , , , , , pax,   

    Novocrania cloning 

    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 19:19 on 2014/11/06 Permalink
    Tags: , , pax,   

    Testing new Novocrania cDNA 

    I’m testing the new cDNA for Novocrania made by Daniel. He made a mix of extractions from different stages and diluted 1:10 and 1:20. I tested with previous genes that worked, pax2/5/8 and wnt11. Set the PCR with a reaction for the 1x, 1:10 and 1:20.

    07/11/2014

    2014-11-07 12.17.42

     
  • Bruno Vellutini 15:32 on 2014/06/22 Permalink
    Tags: , , , pax   

    Novocrania fluorescent in situ for mesoderm 

    Started a Novocrania in situ with engrailed and pax258, two genes that are expressed in the mesoderm. I want to find out the exact location of these genes in the coelomic pouches. And why not a double in situ with both.

    engrailed DNP pax258 DNP engrailed DNP + pax258 DIG

    23/06/14

    Added probes at 10:30.

    25/06/2014

    1st washing day. Everything normal until I was about to dilute the DNP antibody. There was 0.5 µL left and I needed 2 µL! So, passed embryos over to PTw and kept in the fridge until the new anti-dnp hrp arrives…

    01/07/2014

    Washing 5x with PBT and blocking for 1h. Adding the antibodies (anti-dnp 1:100 for engrailed and pax258 and anti-dig 1:250 for pax258 of the double) at 18h in 2mL eppendorf tubes.

    02/07/14

    Washed more than 5 times in PBT for around 10-15 minutes each. Then longer PTw washes, but not as long as 30 minutes. Started developing around 12h with TSA kit with Cy5 for the single fluorescent in situs and Cy3 for the double. Stopped the reaction with 3x 5min washes of detergent solution at 60 °C followed by 3x PTw and standard POD inactivation for the double.

    Single in situs were washed a couple of extra times in PTw and stained with 1:5000 dilution of Sytox Green for 1h30. Embryos were washed in PTw and dehydrated in Methanol. Finally embedded in Murray’s Clear.

    On the fluorescent scope the signal is not clear… there is a lot of background.

    Incubated the double with the anti-dnp antibody overnight.

     
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