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  • Bruno Vellutini 09:53 on 2015/04/06 Permalink
    Tags: , , staining   

    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 18:52 on 2015/02/13 Permalink
    Tags: , , , serotonin, staining, synapsin, , tyrosinated tubulin   

    Brachiopod mesoderm immunostaining 

    Mesoderm and maybe some neurons and muscles. Engrailed antibody second try
    Ttra mixed stages: DAPI / PHALL / Tyros. Tub. 1:500 a594 (mouse) / Synapsin II 1:500 a647 (rabbit) Ttra mixed stages DAPI / PHALL / Serotonin 1:500 a647 (rabbit) / Engrailed 4D9 1:25 a594 (mouse)
    Nano mixed stages: DAPI / PHALL / Tyros. Tub. 1:500 a594 (mouse) / Synapsin II 1:500 a647 (rabbit) Nano mixed stages DAPI / PHALL / Serotonin 1:500 a647 (rabbit) / Engrailed 4D9 1:25 a594 (mouse)
    • 2h permeabilizing in 0.2 % PTx
    • 2h 0.2 % PTx + BSA.
    • 1h 0.2 % PTx + 5 % NGS.
    • Incubated with primary antibodies at 4°C in 0.2% PTx+NGS.

    14/02/15

    • Washed PTx+BSA 3x 5 min.
    • Washed PTx+BSA 4x 30 min.
    • Incubated 1h in PTx+NGS.
    • Added secondaries (alexa 594 mouse and alexa 647 rabbit) in a concentration of 1:200.

    15/02/15

    • Washed PTx+BSA 3x 5min.
    • Washed PBT 5x 10 min.
    • Stained with BODIPY FL and DAPI (1:500) for 2h.
    • Washed out staining with 1x PBS.
    • Overnight at 4 °C.

    16/02/15

    Mounting with Murray Clear went well.

     
  • Bruno Vellutini 19:30 on 2014/04/10 Permalink
    Tags: , , staining   

    Membranipora staining with Phalloidin 647 and Sytox Green in TDE 

    I scanned the U0126 treated embryos with DAPI+Phallacidin in glycerol and the outcome was far from optimal. Penetrance was not great and DAPI is always week… So I decided to try to get a nice morphological staining using TDE, to scan the whole sample without loss; Sytox Green, for strong nuclear staining; and Phalloidin 647, for cell linings and actin. I think this should give a clear picture and nice composition for a morphological stance.

    I selected different stages from cleavage to cyphonautes and added to 2 wells. In one I’m doing the staining above and in the other I’m just mounting them in TDE without phalloidin or sytox. I want to clarify if they have auto-fluorescence at all when embedded in TDE. I suspect they have it on the green.

    Phalloidin + Sytox Nothing, just unstained embryos

    I got 6.25 µL of Phalloidin 647, dried in a tube and diluted in 250 µL of 0.1% PTx with 1:1000 dilution of Sytox Green. Incubated covered for 1h40 and washed 4x 5 min in PBS. Kept the well in the dark at 4 °C.

    11/04/14

    Embed samples in TDE and mount slides to check signal.

    12/04/14

    Confocal?

     
  • Bruno Vellutini 18:00 on 2013/06/05 Permalink
    Tags: , , staining,   

    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 18:00 on 2012/02/26 Permalink
    Tags: , staining   

    Antibody staining for cyphonautes larvae 

    Execute the protocol for antibody staining with Chema to test in cyphonautes larvae of Membranipora. Chosen antibody is phospho-histone which binds to histone in chromatin revealing dividing cells.

    26/02/2012

    Day one of the standard protocol. Incubating overnight with rabbit primary antibody diluted 1:2500 in PTx+NGS.

    27/02/2012

    Day two, washes and incubation with secondary (rabbit) antibody (orange-red AlexaFluor 594) diluted 1:200 in PTx+NGS.

    28/02/2012

    Day three, regular washes and addition of Phallacidin with 1:1000 DAPI. Staining for 1h and mounting in glycerol.

    Mounted 3 slides. 1 is good, 2 has most embryos and most dirt, 3 has only a few embryos. Checked the slides in the fluorescent lamp: DAPI and Phallacidin look great and strong; Phospho-histone signal is widespread in the whole embryo, but seems to be slightly stronger in the apical region.

     
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