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  • 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
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    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.

     
  • 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.

     
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