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  • Bruno Vellutini 16:37 on 2015/08/25 Permalink
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    Membranipora simple staining 

    Just a simple staining of mixed stages of Membranipora using DAPI and Phalloidin 647 for morphology.

    • 3x 30min 0.2% PTx washes.
    • 1x PTx overnight at 4°C.

    26/08/15

    • Staining with Phalloidin and DAPI for 2h.
    • Let embryos in PBS+DAPI 1:1000 at 4°C.

    It failed… weird.

     
  • Bruno Vellutini 12:09 on 2015/08/25 Permalink
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    Membranipora in situ + mapk, a re-try 

    Since I saved some de-glycerolled embryos I’ll repeat the immuno staining with a longer developing time.

    gata b nk2.1
    • Washed 1h in PBT (4x).
    • Washed 3h in 5% NGS (3x).
    • Added anti-mapk antibody 1:100.
    • Let overnight at 4°C.

    26/08/15

    • Washed primary antibody with 3x PBT 5min + 3x 30min.
    • 5% NGS 3x 30 min.
    • Added both anti-mouse-pod/hrp at 1:250 for 4h at room temp.
    • Washed 4x 5min with PBT and let overnight at 4°C.

    27/08/15

    • Wash 1x PBT.
    • Wash 3x TNT buffer.
    • Develop with cy3 for 5min.
    • Wash with PBS (no detergent solution).
    • Embed in TDE.
     
  • Bruno Vellutini 11:47 on 2015/08/18 Permalink
    Tags: , , , gata, , , nk2.1,   

    Membranipora MAPK staining on in situ embryos 

    Selected 5 genes for a MAPK immuno staining to resolve the spatial relationship between MAPK vegetal blastomere, gene expression and the embryonic axes.

    gata_b foxa
    six3/6 evx
    nk2.1

    I started to de-glycerol embryos in hourly washes of PTw around 1200.

    20/08/2015

    • 3x 10min PTx
    • 2x 45 PBT
    • 2x 30min PTx+NGS
    • 1:200 anti-mapk antibody added at 14:00

    21/08/2015

    • Wash primary antibody (afternoon)
    • 3x 5min.
    • 4x 30min.
    • Add secondary antibody anti-mouse pod at 1:250 dilution at 1800.

    22/08/2015

    • 3x 5min PBT washes.
    • 4x 30min PBT washes.
    • Overnight at 4°C in PBT.

    23/08/2015

    • Washed 3x 5min in TNT buffer.
    • Developed in 50 µL TSA + Cy5 fluorochrome for 3 min.
    • Washed 10min in detergent solution at 67°C and another with warm detergent but at room temp to cool down.
    • Washed 2x 5min PBS.
    • TDE series ending with 97% TDE in PBS with DAPI 1:1000.

    24/08/2015

    Mounted slides and checked under scope. There is quite some background with the orange filter for cy3. I cannot distinguish any signal at the posterior vegetal blastomere. Confocal showed that there was no signal from the MAPK antibody. Maybe the longer time in glycerol messed the epitopes? Or the time developing (3min) was not enough…

    DAPI (cyan), MAPK (magenta), in situ (yellow):

     
  • Bruno Vellutini 11:13 on 2015/05/12 Permalink
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    Novocrania wnt1 in situ with longer probe 

    Na wnt1 Tt runx

    Did everything as usual and embryos looked normal.

    13/05/15

    Embryos quite damaged, including Terebratalia. Possible that pipette is not well calibrated?

    Restarted in situ today with extreme gentleness. Added 5 µL protk in 10 mL (instead of 2.5 in 5 µL) using the P20 pipette. Fosfatase wash with 83 °C and added hybe buffer around 1800.

    14/05/15

    Add probe.

     
  • Bruno Vellutini 22:57 on 2015/05/11 Permalink
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    Long probe for Novocrania wnt1 

    Set probe PCR for:

    Na wnt1 BV387: T7 80 ng/µL
    Mm pl10 BV79: SP6 702 ng/µL

    and extracted.

    12/05/15

    Set transcription reaction and precipitation.

    13/05/15

    Finished probes (see values above) and made 1x dilution for wnt1.

     
  • Bruno Vellutini 18:24 on 2015/05/08 Permalink
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    Membranipora in situ and Novocrania wnt1 

    Starting in situ in Membranipora with dorsoventral genes and additional genes that we backgroundish.

    wnt1 dlx bmp2/4 chordin fgf
    nanos vasa piwi1 piwi2 pl10
    Na wnt1

    Standard in situ protocol with 10 min protk. Washes after glycine were slightly shorter and fixing went for 1:30h. Used 83°C for fosfatase step and prehybe at 67°C.

    09/05/15

    Added probes just after synthesis.

    11/05/15

    Novocrania were dissolved. The rest were fine. Continued with hybe washes and added anti-dig-ap.

    12/05/15

    Antibody washes. Plus developing. dlx and piwi1 were stopped after a couple of hours. The remaining were developed overnight at 4 °C.

    13/05/15

    Exchanged the AP and stopped the reaction around 1200. Did ethanol washes and left in 70% glycerol + dapi overnight at room temp.

     
  • Bruno Vellutini 20:09 on 2015/05/06 Permalink
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    Probe PCR Membranipora final genes 

    Probe PCR for bmp/24, chordin, fgf8 and piwi1.

    07/05/15

    Extracted probe PCR gel.

    2015-05-07 11.35.03

    08/05/15

    Bit weird the bmp2/4, but well. Made the transcription reaction for 6:30h and put probes to precipitate.

    09/05/15

    Finish probes.

     
  • Bruno Vellutini 20:07 on 2015/05/06 Permalink
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    Novocrania wnt1 one more time 

    Set PCR with 60°C annealing, 1:45 elongation, 40 cycles and primers at 25mM.

    wnt1 1:10 cDNA wnt1 1:20 cDNA
    pax6 1:10 cDNA pax6 1:20 cDNA

    07/05/15

    2015-05-07 11.35.14

    It worked. Extracted, ligated, transformed and plated.

    08/05/15

    Colony PCR and 2 volonies were fine:

    2015-05-08 17.18.24

    Put these to grow under the IDs:

    T7
    Na wnt1 long BV386
    Na wnt1 long BV387

    09/05/15

    Extract miniprep and set sequencing reaction.

     
  • Bruno Vellutini 18:29 on 2015/04/29 Permalink
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    Membranipora cloning final genes 

    Set PCR with:

    Mm bmp2/4 Mm otx1 Mm chordin Mm fgf8/17/18
    Mm nanos Mm piwi1 Mm piwi2 Mm vasa

    30/04/15

    Ran gel:

    2015-05-01 16.23.27

    Most important genes worked: bmp2/4, chordin and fgf8. piwi1 is bonus. Extract these and set a ligation for 3h. Transformed and plated around 18h.

    01/05/15

    Set colony PCR.

    2015-05-01 16.23.36

    Put colonies to grow.

    02/05/15

    Extracted minipreps, set sequencing PCR and dropped at seq facility.

    BV378 Membranipora membranacea Bmp2/4 T7 ok + SP6
    BV379 Membranipora membranacea Bmp2/4 T7 ok + SP6
    BV380 Membranipora membranacea Chordin T7 ok + SP6
    BV381 Membranipora membranacea Chordin T7 ok + SP6
    BV382 Membranipora membranacea Fgf8/17/18 T7 ok + SP6
    BV383 Membranipora membranacea Fgf8/17/18 T7 ok + SP6
    BV384 Membranipora membranacea Piwi1 T7 ok + SP6
    BV385 Membranipora membranacea Piwi1 T7 ok + SP6

     

     
  • Bruno Vellutini 09:53 on 2015/04/06 Permalink
    Tags: , ,   

    Membranipora MAPK immuno staining 

    I will test whether the MAPK antibody works after in situ hybridization and if it works after U0126 treatment.

    [empty] MAPK after nk2.1 in situ
    MAPK 8h 14°C control MAPK 8h 14°C U0126 treated

    For the in situ well:

    • 3x 10min PTx
    • 2x 45min PBT
    • 2x 30min PTx+NGS

    For the other wells I did longer PTx initial washes (45min total) and shorter PBT and NGS (as in the protocol, 2x 15min PBT, 30min NGS).

    Incubated all at 4°C with 1:200 dilution of anti-mapk (new aliquot from S8).

    07/04/15

    Washed off primary antibody with PBT 3x5min 4x30min. Added secondary anti-mouse pod at 1:250 dilution.

    08/04/15

    Washed secondary with PBT washes as above and developed with 50µL of TSA solution with 1 µL of Cy5 fluorochrome. Embryos were washed 3x5min in PTx and last one for 1h. Results:

    • MAPK immuno staining works after in situ. Signal is weaker and seems to be restricted to the nuclei (cytoplasm signal unclear). The MAPK active cell is located opposite to the expression of nk2.1, which is anterior/ventral. Thus, it seems likely that the vegetal cell with MAPK activity is at the posterior/dorsal side. Plan appropriate in situs to confirm this result.
    • Controls worked ok.
    • U0126 10µM treated embryos showed no detectable MAPK activity. One embryo might had a brighter nuclei, but I need to confirm this under the confocal. Another issue is that these samples had the membrane. MAPK antibody works fine through the membrane, but maybe is better to remove them to have a cleaner image and avoid doubt.

    There was some extra background, I’ll wash the embryos with detergent solution to remove some.

    09/04/15

    Made 30min at 67°C detergent washes (total 3x 10min). Background improved a bit, but nothing extreme.

    Also did a Murray’s Clear preparation and they get completely transparent. However, the morphology is not quite optimal and embryos get fragile. Not sure if worth it.

     
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