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  • Bruno Vellutini 11:47 on 2015/08/18 Permalink
    Tags: , , , gata, , , nk2.1, six3/6   

    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 19:30 on 2012/11/16 Permalink
    Tags: brachyury, , , , , , , six3/6,   

    Lineus ruber and Membranipora in situ 

    Starting the first in situ in the nemertean Lineus ruber and trying different genes in Membranipora after establishing the ProtK timing. Selected genes are:

    L. ruber foxa six3/6 cdx gsc
    M. membranacea bra gsc piwi1 vasa

    All wells treated with 10 min in 1x protk concentration (0,01 mg/mL).

    17/11/12

    Added probes.

    19 and 20/11/12

    Washing days, normal activity. Samples stayed in blocking buffer for 1h30 (instead of 1h) due to epic foosball match.

    21/11/12

    Began developing at 11h. Twenty minutes later Mm bra cyphonautes shell were already completely blue… It seems that mucus cells in Lineus juveniles are being stained. No real signal coming up, except in six3/6, maybe. Mm piwi1 and vasa look like to be working, something is appearing. Exchanged AP once and let it at 4°C.

    22/11/12

    Changed the AP of Lineus twice. Some signal kind of visible below the epidermis, nothing in the embryos. Stopped Membranipora development.

    23/11/12

    Patterns in Lineus more visible and apparently real signal. foxa expressed in the gut region, six3/6 in the brain/anterior, cdx in the posterior tip (not in the epidermis), and gsc in the anterior mouth region. Let developing one more day at 4°C.

    25/11/12

    Stopped reaction. Background began to come up, but I could check the expression more clearly. For example, foxa is detected in the mouth region and cdx in the anus. Now what needs to be done is remove the mucus cells with cystein before fixation.

    26/11/12

    Restart development of Lineus to see if the signal get stronger in the juveniles and something appears in the embryos.

    29/11/12

    Finally stopped the development. Will do ethanol washes tomorrow.

     
  • Bruno Vellutini 19:27 on 2012/11/13 Permalink
    Tags: , , , , , , , , , six3/6, ,   

    Probe synthesis for Lineus et al. 

    Started a probe pcr for synthesizing the following probes of Lineus ruber, Priapulus caudatus, and Terebratalia transversa:

    Species Gene ID Probe Kit
    Lineus ruber foxa BV196 SP6
    Lineus ruber six3/6 BV199 SP6
    Lineus ruber cdx BV204 SP6
    Lineus ruber gsc BV205 T7
    Priapulus caudatus tdr BV34 T7
    Priapulus caudatus mael BV35 SP6
    Priapulus caudatus gmcl BV183 T7
    Terebratalia transversa runx2 BV166 SP6

    13/11/12

    Run gel.

    14/11/12

    Dna extraction.

    15/11/12

    Transcription reaction + precipitation.

    16/11/12

    Specs:

    Lr foxa 688,23
    Lr six3/6 383,21
    Lr cdx 303,11
    Lr gsc 501,31
    Pc tdr 3853,39
    Pc mael 97,89
    Pc gmcl 2287,30
    Tt runx2 345,39

     
  • Bruno Vellutini 19:01 on 2012/10/29 Permalink
    Tags: , , , , , , , six3/6   

    Lineus ruber first cloning 

    Trying to clone 6 genes: six 3/6, caudal, goosecoid, foxa, foxq2, and fgf8/17/18. PCR was not so successful, only foxa and six3/6 worked:

    30/10/12

    Set ligation.

    31/10/12

    Transformed and plated.

    01/11/12

    Colony PCR for both genes:

    03/11/12

    Minipreps.

    Colony T7
    foxa 4 BV196
    foxa 5 BV197
    foxa 6 BV198
    six3/6 6 BV199
    six3/6 7 BV200
    six3/6 8 BV201

     
  • Bruno Vellutini 12:29 on 2012/07/23 Permalink
    Tags: , , , , , , , , , six3/6, ,   

    PCR mostly stem/germ cell genes of Terebratalia 

    Setting up a pcr with gradient (63 +- 3 °C) at 12h:

    Gene Primers Tm mean
    Mm Six3/6 64.10
    Tt Dicer1b 61.60
    Tt Runx 63.15
    Tt Mael 65.15
    Tt Piwia 65.55
    Tt Tudor1 61.95
    Tt Ago 62.95
    Tt PL10 63.10
    Tt Pum 65.35

    Extraction and ligation overnight at 4°C, except for MAEL.

    24/07/12

    Transformation and plating of colonies.

    25/07/12

    PL10 and Pumilio had very few colonies on the plate (insert numbers) and together with Piwia they did not produce any positive colony.

    27/07/12

    Put positive colonies to grow for minipreps.

    BV162 Terebratalia transversa Dicer1b
    BV163 Terebratalia transversa Dicer1b
    BV164 Terebratalia transversa Runx2
    BV165 Terebratalia transversa Runx2
    BV166 Terebratalia transversa Runx2
    BV167 Terebratalia transversa Tudor1
    BV168 Terebratalia transversa Argonaute
    BV169 Terebratalia transversa Argonaute
    BV170 Terebratalia transversa Argonaute
    BV171 Terebratalia transversa NK6
    BV172 Terebratalia transversa NK6
    BV173 Terebratalia transversa NK6
    BV174 Terebratalia transversa Wnt7
    BV175 Terebratalia transversa Wnt7
    BV176 Terebratalia transversa Wnt7

     
  • Bruno Vellutini 12:30 on 2012/04/26 Permalink
    Tags: , , , six3/6   

    Primers Tt en, Tt dlx, and Mm six3/6 just arrived.

     
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