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  • Bruno Vellutini 11:47 on 2015/08/18 Permalink
    Tags: , even-skipped, , 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 20:10 on 2014/04/29 Permalink
    Tags: even-skipped, ,   

    Mounted and took some pictures of Membranipora in situs Chema did. Pattern looks sharp and with no background in the early stages. evx is restricted to one ectodermal cell during early gastrulation and 2 large lateral vegetal cells get activated later and it ends in the midgut and hindgut in early larva.

    20140429_192744

     
  • Bruno Vellutini 12:44 on 2013/02/13 Permalink
    Tags: , , , even-skipped, ,   

    Probe synthesis for old and new Terebratalia segmentation genes 

    Transcription reaction set for at 10h30 and precipitation at 17h00:

    ID GENE ENZYME UTP ng/µL
    BV208 en SP6 DIG 135
    BV208 en SP6 DNP 55
    BV209 dlx SP6 DIG 357
    BV211 wnt4 SP6 DIG 351
    BV218 wnt11 SP6 DIG 1234
    BV210 cdx T7 DIG 1450
    BV212 wnt8 T7 DIG 129
    BV213 eve T7 DIG 2269

    14/02/13

    Pellet washing and specs taken, see above. en was the least concentrated one, specially with DNP utp. Will need to re-do it to get more probe.

     
  • Bruno Vellutini 17:17 on 2013/02/07 Permalink
    Tags: , , , , even-skipped, , ,   

    Transformation of plasmids for Terebratalia new probes 

    Some of the genes cloned by Yale and Andi will be used for double in situs. We have no probes and there is a limited amount of plasmid solution. So I’m transforming the plasmids again to make more minipreps (and then probes). The genes are:

    Gene ID Kit
    en TTR54 SP6
    dlx Tt-dlx-L2 SP6
    cdx TTR16 T7
    wnt4 TTR164 SP6
    wnt8 TTR104 T7
    eve Tt-eve-L2 T7

    I used 1 µL of plasmids for the transformation and diluted the bacteria solution prior to plating by 1:100 (1µL bacteria: 99µL LB at 37°C). I then plated 50 µL of the 1:100 solution at 16h30.

    08/02/13

    Perfect number of colonies!

    2013-02-08 17.42.06

    09/02/13

    T7
    en BV167
    dlx BV168
    cdx BV169
    wnt4 BV170
    wnt8 BV171
    eve BV172

    10/02/13

    Extracted the plasmids and started the probe PCR.

    11/02/13

    Probe PCR extracted.

    2013-02-11 15.07.35

     
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