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

    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 18:52 on 2015/02/13 Permalink
    Tags: , engrailed, , 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 12:49 on 2015/01/30 Permalink
    Tags: engrailed, , , , , , , ,   

    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: engrailed, , , , , , ,   

    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: , engrailed, , , , , , , 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 15:32 on 2014/06/22 Permalink
    Tags: engrailed, , ,   

    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.

     
  • Bruno Vellutini 16:03 on 2014/06/06 Permalink
    Tags: , engrailed, , , , ,   

    Brachiopod in situ 

    In situ for additional Terebratalia frizzleds and repeating some Novocrania for better pictures.

    Nano engrailed wnt1 wnt5 dlx pax258
    Ttra frizzled 5/8 frizzled 7 dcr1b tdr1 ago

    Started in situ with standard procedures. 8 min protk for Novocrania and 10 min for Terebratalia.

    07/06/14

    Added probes, embryos look good.

    09/06/14

    Washing day. Everything went as usual and put antibody around 18h.

    10/06/14

    Washed with PBT several times berween 10-15 min. Then PTw 30 min washes. Started developing.

    Stopped Nano dlx after 2h30min because it was done, as well as Ttra fz5/8. The rest continued developing. fz7 got pinkish really fast

    11/06/14

    fz7 and ago got really dark. I don’t know if it is background or signal, even though it looks like there is real signal within the background. I stopped them as well as engrailed and pax258, which look better than the last in situ.

    12/06/14

    Stopped wnt1, wnt5 dcr1b, tdr1. Novocrania samples are looking dark ugly already…

    13/06/14

    Trying to clear out some of the background from fz7 and ago… letting in ethanol for many hours.

     
  • Bruno Vellutini 08:00 on 2014/04/05 Permalink
    Tags: , engrailed, , , ,   

    Terebratalia Azakenpaullone treated in situ wnt1 and engrailed 

    Starting in situ with azakenpaullone-treated Terebratalia embryos with wnt1.

    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-

    Prepared fresh PTw and went up to hybe step. Then split samples for wnt1 and engrailed (30 wells in total). Wnt1 probe will be fine, but engrailed will be much less concentrated, probably around 0.1 ng/µL. Unfortunately this is what I had in stock.

    06/04/14

    I fell face to the ground and could not go to the lab. Chema put my 2 plates in the freezer.

    07/04/14

    I put the plates back at 62 °C for 5h. I diluted wnt1 probes and added the usual 500 µL volume to the wells. For the engrailed probe I had to dilute it to 0.8 ng/µL and use only 250 µL of volume… but well.

    09/04/14

    Washing day. Everything went normally. I added the antibody around 17:30.

    10/04/14

    Washed 2 x 15 min and 1x 1h with PBT and then 1x 15 min. Finally 5x 30 min with PTw. Left the plates in the cold room.

    11/04/14

    Developing day. Ran for 5h and some signal is appearing; stronger in the treated embryos than in the controls. Exchanged the AP substrate at 17:30 and put in the cold room.

    12/04/14

    Most of the treated embryos have a nice signal. Controls are surprisingly faint, though. I developed through the whole day and kept in the cold room overnight. Probably stopping tomorrow some wells.

    13/04/14

    Stopped the reactions in the treated samples with 3 washes of AP without magnesium chloride. I’m still developing the controls.

    14/04/14

    Still developing controls. Stopped wnt1, but not engrailed.

    15/04/14

    Exchanged again the AP. It looks much better now, signal is stronger. I did not filter the AP this time to see if I get crystals.

    Developed for 8h and stopped the reaction on the last wells.

    16/04/14

    Ethanol washes upt to glycerol.

     
  • Bruno Vellutini 14:34 on 2014/01/16 Permalink
    Tags: engrailed, , ,   

    Vivo-Morpholino next with pronase and electroporation 

    Considering my last observations of the vivo-morpholinos I’m setting up a new experiment using a lower concentration of pronase, electroporating twice (because I could not figure out how to do longer pulses), and properly washing out the pronase after 1h. Default settings:

    Vivo-Morpholino: 25 µM
    Pronase: 5 µg/mL
    Washing after: 1h
    Electroporation: 50 V, 25 µF, 1000 resistance
    Actual pulses:

    CTRL FSW CTRL Pronase EN TB Pronase EN SB Pronase WNT1 TB Pronase WNT1 SB Pronase
    CTRL Electroporation EN TB Electroporation EN SB Electroporation WNT1 TB Electroporation WNT1 SB Electroporation

     
  • Bruno Vellutini 20:17 on 2014/01/15 Permalink
    Tags: engrailed, , ,   

    Terebratalia Vivo-Morpholino engrailed/wnt1 with permeabilization 

    Use 25 µM concentration for vivo-morpholinos.

    CTRL FSW
    CTRL CFSW 1h Ttra_en_TB_vMO 25 µM Ttra_en_SBe2i2_vMO 25 µM Ttra_wnt1_TB_vMO 25 µM Ttra_wnt1_SBe2i2_vMO 25 µM
    CTRL Pronase 10 µg/mL 1h Ttra_en_TB_vMO 25 µM Ttra_en_SBe2i2_vMO 25 µM Ttra_wnt1_TB_vMO 25 µM Ttra_wnt1_SBe2i2_vMO 25 µM
    CTRL Electroporation in CFSW Ttra_en_TB_vMO 25 µM Ttra_en_SBe2i2_vMO 25 µM Ttra_wnt1_TB_vMO 25 µM Ttra_wnt1_SBe2i2_vMO 25 µM

    Electroporation setup

    BioRad Gene Pulser 50V, 25 µF, resistance set to infinity.

    [photo]

    ms
    ctrl 1.1
    en tb 1.0
    en sb 1.1
    wnt1 tb 1.2
    wnt1 sb 0.9

    16/01/14

    • Calcium-free seawater look normal trilobed.
    • Pronase treatment embryos failed to gastrulate, including the control.
    • Electroporation look normal except for wnt1 sb which seem either delayed or did not manage to close the blastopore.
     
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