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  • Bruno Vellutini 09:50 on 2015/04/05 Permalink
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    Membranipora in situ with mesodermal genes 

    First in situ with mesodermal genes of Membranipora plus some extras. Will add a control with Chema’s probe and do only early stages until late gastrula so I don’t have to worry with cyphonautes larvae.

    foxa foxc foxd foxf
    mef2 noggin2 eya (long) mprx
    fhl mox pax6 wnt4

    ProtK lasted 10min30s but washed with 800/500 µL. Otherwise standard protocol. They stayed longer (30min) in the first hybe buffer until the oven was free. Pre-hybe in the oven at 67 °C.

    06/04/15

    Put probes.

    08/04/15

    Standard washes as usual. Stayed 1h30 in 1x blocking before I added the antibodies.

    09/04/15

    Wash off secondary antibody. Started developing. Most genes did not show any clear staining except for pax6 in mid gastrula and foxa. Background was much better than the previous in situ, but not completely absent. Maybe try an even higher temperature?

    10/04/15

    Now there is visible signal in all fox genes: early single cell expression in early gastrula and in the anterior muscles of the late gastrula. eya expression also came up in some internal cells. Other genes have no clear signal. I stopped all the reactions by the end of the day.

    11/04/15

    Ethanol washes and placed in 70% glycerol with DAPI.

    12/04/15

    I’ll mount some slides and check the patterns.

     
  • Bruno Vellutini 18:45 on 2015/03/30 Permalink
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    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 18:16 on 2015/03/28 Permalink
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    Probe synthesis Membranipora mesodermal genes 

    Set probe PCR for the first batch of genes published by the sequencing facility.

    id species gene enzyme ng/µL comment
    BV359 Membranipora membranacea FoxC SP6 1020
    BV361 Membranipora membranacea FoxD SP6 445
    BV362 Membranipora membranacea FoxF SP6  545
    BV363 Membranipora membranacea Mef2 SP6  449
    BV365 Membranipora membranacea Noggin2 SP6  988
    BV367 Membranipora membranacea Eya SP6  826 short isoform
    BV368 Membranipora membranacea Eya SP6  1271 long isoform
    BV370 Membranipora membranacea mPRX SP6  620
    BV372 Membranipora membranacea FHL SP6  321
    BV374 Membranipora membranacea Pax6 SP6  1072
    BV375 Membranipora membranacea Mox T7  1066
    BV376 Membranipora membranacea Wnt4 SP6  1647

    30/03/2015

    Ran gel and extracted probe pcr.

    2015-03-30 17.37.26

    31/03/2015

    Ran and extracted probe pcr for the second batch of sequences.

    2015-03-31 12.19.30

    2015-03-31 12.19.22

    02/04/2015

    Started riboprobe synthesis reaction. Ran for 6.5h and set to precipitate with 5µL of LiCl2.

    03/04/2015

    Precipitate and finish probes. Check above for values.

     
  • Bruno Vellutini 18:39 on 2015/03/23 Permalink
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    Cloning Membranipora mesodermal genes 

    Cloning mesodermal genes in Membranipora: wnt4, pax6, noggin, mox, paraxis, foxf, mprx, nog2, mef2, fhl, foxc, foxd, eya. Nano wnt1 longer probe 2try.

    24/03/15

    2015-03-23 19.57.29

    25/03/15

    2015-03-25 16.45.11

    26/03/15

    Extract minipreps and sequencing. Done. BV359 to BV377 with T7 primer.

    BV359 Membranipora membranacea FoxC T7 ok + SP6
    BV360 Membranipora membranacea FoxC T7 ok + SP6
    BV361 Membranipora membranacea FoxD T7 ok + SP6
    BV362 Membranipora membranacea FoxF T7 ok + SP6
    BV363 Membranipora membranacea Mef2 T7 ok + SP6
    BV364 Membranipora membranacea Mef2 T7 ok + SP6
    BV365 Membranipora membranacea Noggin2 T7 ok + SP6
    BV366 Membranipora membranacea Noggin2 T7 ok T7
    BV367 Membranipora membranacea Eya T7 ok + SP6 short isoform
    BV368 Membranipora membranacea Eya T7 ok + SP6 long isoform
    BV369 Membranipora membranacea Eya T7 ok T7
    BV370 Membranipora membranacea mPRX T7 ok + SP6
    BV371 Membranipora membranacea mPRX T7 ok T7
    BV372 Membranipora membranacea FHL T7 ok + SP6
    BV373 Membranipora membranacea Pax6 T7 ok T7
    BV374 Membranipora membranacea Pax6 T7 ok + SP6
    BV375 Membranipora membranacea Mox T7 ok T7
    BV376 Membranipora membranacea Wnt4 T7 ok + SP6
    BV377 Membranipora membranacea Wnt4 T7 ok + SP6

     
  • Bruno Vellutini 18:01 on 2015/02/27 Permalink
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    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
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    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, , 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
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    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
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    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
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    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.

     
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