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  • Bruno Vellutini 09:50 on 2015/04/05 Permalink
    Tags: eya, , , , , , , , , , , , ,   

    Membranipora in situ with mesodermal genes 

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    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.


    Put probes.


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


    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?


    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.


    Ethanol washes and placed in 70% glycerol with DAPI.


    I’ll mount some slides and check the patterns.

  • Bruno Vellutini 18:16 on 2015/03/28 Permalink
    Tags: eya, , , , , , , , , , ,   

    Probe synthesis Membranipora mesodermal genes 

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    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


    Ran gel and extracted probe pcr.

    2015-03-30 17.37.26


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

    2015-03-31 12.19.30

    2015-03-31 12.19.22


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


    Precipitate and finish probes. Check above for values.

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