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  • Bruno Vellutini 17:03 on 2013/10/27 Permalink
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    Novocrania in situ 3rd try 

    Third try with engrailed, wnt1, and wnt5. This time using half-concentrated 5 min ProteinaseK treatment. They should not get damaged!

    • Fixed for 1h25min.
    • Washed normally and incubated in pre-hybe.

    28/10/13

    Added probes at 11h.

    30/10/13

    Washing day embryos looking good.

    31/10/13

    Washed out antibody with long 30 min washes.

    01/11/13

    Started developing. Engrailed began to appear after 2hs, but nothing in the wnts. I exchanged the AP substrate every 2 hours. At 18h I transferred to new wells because crystals were forming. At 19h30 I exchanged the AP and put them in the cold room.

    02/11/13

    Stopped engrailed and continued developing the wnts during the day. No signal came up except for background. Paused in AP without magnesium.

    04/11/13

    Restarted the wnts to see if something comes up…

    05/11/13

    Stopped wnts… Ethanol washes and added glycerol+DAPI overnight in cold room.

    06/11/13

    Mount slides. Engrailed has some shitty background… Remember next time to stop before overnight.

     
  • Bruno Vellutini 16:29 on 2013/10/21 Permalink
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    Priapulus profiling in situ matching 

    I got some in situ pictures from Priapulus and  tried to match them to the staged expression levels data. They are congruent more or less, although it is not so straightforward since stage pictures is not complete (early/late missing) and embryos are not exactly the specific rna stages.

    However, I can see gastrulation as a major burst of expression for most of the genes we have the in situs.

    bcatenin brachyury cdx evx fgf8 foxa1 foxq2 fz58 gata456 gsc

     
  • Bruno Vellutini 13:20 on 2013/10/21 Permalink
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    Terebratalia additional double in situs 

    Started Terebratalia double in situs with the following selected probes. Everything looks ok.

    wnt1 (DNP) + wnt4 (DIG) en (DNP) + wnt7 (DIG)
    wnt1 (DNP) + wnt8 (DIG) en (DNP) + pax258 (DIG)

    22/10/13

    • Added probes to Terebratalia doubles.

    24/10/13

    • Terebratalia doubles washing day.
    • Add Terebratalia first antibody anti-dig-pod 1:250.

    25/10/13

    • Terebratalia doubles wash off first antibody.

    27/10/13

    • Terebratalia doubles develop first probe and inactivate.
    • Terebratalia doubles incubate with second antibody.

    28/10/13

    • Terebratalia doubles wash off second antibody.

    29/10/13

    • Terebratalia doubles develop second probe.
    • Terebratalia doubles stain with phallacidin.
    • Terebratalia doubles embed in TDE+DAPI.

    30/10/13

    • Mount Terebratalia doubles.

    31/10/13

    • Confocal slot for Terebratalia doubles.

    01/11/13

    Here are some scans.

     
  • Bruno Vellutini 09:55 on 2013/10/15 Permalink
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    Novocrania in situ en wnt1 wnt5 2nd try 

    Second chance on the Novocrania in situ which failed last time. This time I’ll do 7 minutes of ProtK and wash extensively with glycine to stop the reaction.

    3 embryos of each stage: radial, bilateral, early larva, and larva.

    engrailed wnt1 wnt5
    • Initially in MeOH.
    • 1x 5min 60% MeOH 40% PTw.
    • 1x 5 min 30% MeOH 70% PTw.
    • 4x 5 min PTw.
    • ProteinaseK for 7 min.
    • Stopped exactly at 7 min with 900 µL of glycine.
    • 1x 5 min glycine.
    • 1x 5 min 1% TEA.
    • 1x 5 min 1% TEA + Ac. An. –> embryos stick a bit to bottom.
    • Added 100 µL of Ac An. embryos continue to stick.
    • Washed 2x PTw 900 µL.
    • Fixed in 4% FA for 1h25.
    • Washed 3x PTw.
    • 1x 10 min PTw at 80 °C.
    • 1x 5 min PTw.
    • 1x 10 min Hybe Buffer at RT.
    • Exchanged hybe and incubate at 62 °C.

    16/10/13

    • Added probes.
    • Embryos were still looking ok, although somewhat transparent.

    18/10/13

    • Larvae look damaged and may fall apart. Saw a bilateral missing a piece… Otherwise it looks better than last time. I think the time or concentration of ProtK (newly opened) really needs to be decreased!
    • 10 min and 40 min washes with hybe wash.
    • Series of 30 min hybe to 2x SSC.
    • 3x 20 min 0.2X SSC + 0.1% Tween-20.
    • Grade-series of 0.2X SSC to PTw 10 min each.
    • 5x 5 min PBT.
    • Incubate 1h with 1x blocking buffer made with the last 500 µL of 5x blocking buffer from the fridge and newly made maleic acid buffer.
    • Exchanged for 1:5000 Anti-DIG-AP in blocking buffer and incubated overnight in cold room.

    19/10/13

    Embryos still look integer although larvae are damaged.

    • Started washing with PBT at 13h.

    20/10/13

    • Began to develop at 16h.
    • Faint band of engrailed at 19h.
    • Noticed some precipitated when exchanging NBT/BCIP, maybe old BCIP (it is running out)?
    • Let overnight in the cold room.

    21/10/13

    • Checked at 11h and the engrailed stripe was more prominent; nothing yet in the others.
    • There are crystals forming in the bottom :( and some clear precipitate floating around…
    • I exchanged the AP substrate and let it go under RT.
    • Exchanged again after 2h.
    • Final exchange and cold room at 17h30. Maybe there was a bit of background coming up in engrailed… not sure so I let it overnight and will stop tomorrow.

    22/10/13

    • engrailed well began to show some background and the others were looking bad with crystals packing up. So I stopped them all.
    • AP -magnesium 2x.
    • Ethanol washes.
    • 70% Glycerol exchanged once for glycerol with 1:5000 DAPI.
    • I restarted the wnts because I felt bad. Let them develop overnight.

    23/10/13

    • Stopped wnts and did alcohol series.
    • Mounted and took photos of engrailed.
     
  • Bruno Vellutini 18:11 on 2013/10/13 Permalink
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    Comparing Murray Clear and TDE 

    I want to compare mounting with Murray Clear and TDE and decide what is the best option for analysing the morphology of Terebratalia. Murray seems to clear the yolk while TDE only optimizes the refraction index of the immersion liquid. For this I got Terebratalia late larvae and will stain them with phallacidin and propidium iodide.

    • Got Terebratalia late larvae (6 and 7 days).
    • 3x quick washes in 0.2% PTx.
    • 2x 5 min 0.2% PTx.
    • 2x 10 min PBT.
    • Incubated 2h using 1.25 U in 250 µL of Phallacidin (6.25 µL of stock) and 1:500 dilution of propidium iodide.
    • Mount in Murray Clear and TDE.
    • Check staining in the confocal.

    Murray Clear + Phall/PI

    MAX_Ttra TDE vs Murray - Phall-PI.lif - Series007 Murray-3

    Murray Clear embryos shrank a lot and the morphology looks quite altered. Other than that, embryos are clearer and phallacidin works.

    TDE + Phall/PI

    MAX_Ttra TDE vs Murray - Phall-PI.lif - Series015 TDE-3

    TDE is imcompatible with phallacidin and we get a artefact general cytoplasm staining. However, the morphology is quite preserved and you can scan the bottom regions of the sample well.

     
  • Bruno Vellutini 18:51 on 2013/10/11 Permalink
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    Terebratalia engrailed immuno staining 

    I will try using the engrailed/invected 4D9 antibody in Terebratalia.

    • Get radial, asym., bilateral, and trilobed larvae (used mixed stages from Kristineberg samples).
    • Permeabilize with 0.2% PTx (5x 5 min, 1x 30 min).
    • 2x 15 min PBT.
    • 1x 30 min 5% NGS.
    • 17h45: incubate with primary on a tube with 100 µL and dilution of 1:20.

    13/10/13

    • Wash out primary antibody along the day (3x 5 min PBT, 4x 30 min PBT, 1x 30 min PTx+NGS).
    • Incubate with secondary anti-mouse-pod 1:250 overnight.

    14/10/13

    • Washed 3x 5 min in PBT.
    • 4x 30 min PBT.
    • Develop with TSA and Cy5 fluorochrome (49 µL diluent + 1 µL fluorochrome).
    • Stop the reaction with 1x detergent solution at 60 °C and 2x at RT.
    • Washed 3x 5 min in 0.2% PTx.
    • Stained with phallacidin for 2h.
    • Washed 3x 5 min in PBS.
    • Grading series with TDE and mounted in 97% TDE with DAPI.

    15/10/13

    Confocal time. Engrailed antibody is indeed not working. I could try a higher concentration, maybe..

    Ttra DAPI-Phall TDE 1.lif - Series018 Ttra DAPI-Phall TDE 1.lif - Series021 Ttra DAPI-Phall TDE 1.lif - Series024 Ttra DAPI-Phall TDE 1.lif - Series027

     
  • Bruno Vellutini 10:52 on 2013/10/10 Permalink
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    Priapulus profiling initial 

    Screenshot 2013-10-10 09.38.45

    I ran a basic RSEM analysis during the weekend to calculate the expression levels for Priapulus stages. Now I’m following the stepsfor the visualization of the results. The photo above shows the expression levels of the same for different stages. I’m converting WIG files exported with RSEM to TDF to be visualized in IGV.

     
  • Bruno Vellutini 15:33 on 2013/10/01 Permalink
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    • Novocrania pulse & chase EdU 10 min. Fixing at 0, 3, 6, 9h.
    • Also EdU 20 min for in situ with 48h and 54 hours.
     
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