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  • Bruno Vellutini 10:30 on 2013/06/25 Permalink
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    mamed2013_bcv

     
  • Bruno Vellutini 13:04 on 2013/06/19 Permalink
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    Isodiametra pulchra neuropeptide extraction 

    Extracted neuropeptides off the acoel Isodiametra pulchra using Gaspar Jekely protocol. Instead of using a mesh I gave a quick wash on distilled H2O, spin down the worms, then added 250 µL of extraction buffer and drilled them. After I added 350 µL more and continued with the protocol (20 min spin at 4°C). After spinning there was remnants of worms in the bottom, so I just took the clear supernatant and transferred to a new tube.

     
  • Bruno Vellutini 18:00 on 2013/06/05 Permalink
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    Antibody staining with Anti-MAPK 

    Try Anti-MAPK antibody in Membranipora cleavages, Terebratalia gastrula, and Lineus cleavage and also Tyrosinated tubulin in Terebratalia larva as control.

    We split the sample to develop with DAB/HRP protocol and fluorescent.

    Anti-MAPK

    Membranipora

    Cleavages

    Anti-MAPK

    Terebratalia

    Gastrula

    Anti-MAPK

    Lineus viridis

    Cleavage

    Tyrosinated tubulin

    Terebratalia

    Larvae

    Primary antibodies

    Monoclonal Anti-MAP Kinase, Activated (Diphosphorylated ERK-1&2) antibody produced in mouse
    Suggested dilution: 1:1000 (~0.5-1 μg/mL, see specs).
    Observations: @Henry2008 used 1:200 (~30 µg/mL) with a different antibody; @Koop2007 used a final concentration of ~3 µg/mL. @Amiel2013 used 1:200, but does not specify which antibody was used.
    Final concentration we used: 30 µg/mL.

    Tyrosinated Tubulin (Mouse) 1:250.

    Secondary antibodies

    • Anti-Mouse (Goat) HRP + POD (Jackson) → 1:250 each! (1 µL for each of the tubes we have)
    • Anti-Mouse (Goat) AlexaFluor 594 → 1:250.

    Protocol

    We always used 0.3% PTx except for Membranipora (0.1% PTx) with 500 µL washes.

    1st day

    • 5x 5 min washes in PTx.
    • 1x 30 min in PTx.
    • 1x 1h in PTx.
    • 2x 15 min in PTx+BSA.
    • 1x 30 min in PTx+NGS.

    Incubated with Anti-MAPK 30 µg/mL and Tyrosinated tubulin 1:250 at 17h30.

    2nd day 06/06/13

    • 3x 5 min PTx+BSA
    • 5x 30 min PTx+BSA
    • 1x 30 min PTx+NGS

    Samples were split to 2 dishes and incubated with secondary antibodies as described above.

    3rd day 07/06/13

    DAB-HRP reaction (DAB powder borrowed from S4).

    1. Used similar buffer as for in situ: TBS = 20 mM Tris, 500 mM NaCl, pH 7.5.
    2. Dissolve 50 mg DAB in 100 ml TBS. Add 10 μL 30% H2O2. Make this solution just before use.
    3. Remove PTx+BSA and add DAB+H2O2 solution to well. Staining comes immediately, we left for 6 min and it became overdeveloped in the larvae. In Membranipora it was fine.
    4. Stopped the reaction with 2x 5 min washes of PBS (protocol recommends distilled H2O).

    Terebratalia larvae and Membranipora 32 cell embryo worked! We lost fluorescent control and Lineus and Terebratalia gastrula showed no staining, except for what it looked to be unfertilized eggs from Terebratalia.

    Mmem_DAPI Mmem_MAPK

     
  • Bruno Vellutini 13:33 on 2013/06/01 Permalink
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    Beginning a double in situ with en dnp… 

    Beginning a double in situ with:

    en (dnp) + gli (dig)
    en (dnp) + fgf8/17/18 (dig)

    02/06/13

    Added probes.

    04/06/13

    First washing day normal. Washes followed at times, but second 0.2x SSC took 40 instead of 20 min. Added Anti-Dig-POD at 1:250 concentration.

    05/06/13

    Second washing day normal.

    06/06/13

    Developed first probe. CAOS after detergent wash, during subsequent PTw washes the tape glued to the cover and FLIPPED the 4-well dish… I recovered most embryos and many remained in their wells, but for sure there has been some mixing. I added the recovered embryos to a third well.

    Apart from that the first probe works. And incubated with Anti-DNP at 1:100.

    07/06/13

    Regular washes.

    08/06/13

    Developed second probe and let it in PTw in the cold room until 12/06/13.

    12/06/13

    DAPI staining for 20 min, one wash of PTw and glycerol overnight.

     
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