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  • Bruno Vellutini 12:09 on 2015/08/25 Permalink
    Tags: mapk,   

    Membranipora in situ + mapk, a re-try 

    Since I saved some de-glycerolled embryos I’ll repeat the immuno staining with a longer developing time.

    gata b nk2.1
    • Washed 1h in PBT (4x).
    • Washed 3h in 5% NGS (3x).
    • Added anti-mapk antibody 1:100.
    • Let overnight at 4°C.


    • Washed primary antibody with 3x PBT 5min + 3x 30min.
    • 5% NGS 3x 30 min.
    • Added both anti-mouse-pod/hrp at 1:250 for 4h at room temp.
    • Washed 4x 5min with PBT and let overnight at 4°C.


    • Wash 1x PBT.
    • Wash 3x TNT buffer.
    • Develop with cy3 for 5min.
    • Wash with PBS (no detergent solution).
    • Embed in TDE.
  • Bruno Vellutini 11:47 on 2015/08/18 Permalink
    Tags: , , , gata, mapk, , 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

    I started to de-glycerol embryos in hourly washes of PTw around 1200.


    • 3x 10min PTx
    • 2x 45 PBT
    • 2x 30min PTx+NGS
    • 1:200 anti-mapk antibody added at 14:00


    • Wash primary antibody (afternoon)
    • 3x 5min.
    • 4x 30min.
    • Add secondary antibody anti-mouse pod at 1:250 dilution at 1800.


    • 3x 5min PBT washes.
    • 4x 30min PBT washes.
    • Overnight at 4°C in PBT.


    • 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.


    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 09:53 on 2015/04/06 Permalink
    Tags: mapk, ,   

    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).


    Washed off primary antibody with PBT 3x5min 4x30min. Added secondary anti-mouse pod at 1:250 dilution.


    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.


    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 13:06 on 2014/09/23 Permalink
    Tags: , , mapk, ,   

    4D with U0126 Membranipora 

    15 °C, 15 µM

    BCV_Mm_2014_09_23 (old Mmem1_23_09_2014)

  • Bruno Vellutini 13:00 on 2014/09/20 Permalink
    Tags: , , mapk, ,   

    4D with U0126 Membranipora 

    BCV_Mm_2014_09_20 (old Mmem1_20_09_2014)

    10 µM, 15 °C

  • Bruno Vellutini 15:34 on 2014/09/19 Permalink
    Tags: , mapk, ,   

    Membranipora U0126 inhibitor time windows 

    I set the U0126 inhibitor 10 µM with Membranipora at 15 °C. I am washing every 2 hours after 1h30 hours after activation as shown below (2x, for 24h and 48h fixation).

    • Activated: 1100
    • Inhibited: 1230
    • 1st wash: 1430
    • 2nd wash: 1630
    • 3rd wash: 1830
    DMSO 3.5 hpa
    5.5 hpa 7.5 hpa


    Fixed 24h.


    Fix 48h.

  • Bruno Vellutini 17:07 on 2014/09/18 Permalink
    Tags: , , mapk, ,   

    Membranipora 4D U0126 10 µM 

    Started a recording BCV_Mm_2014_09_18 (old Mmem1_18_09_2014) with Membranipora in 10 µM U0126 MEK inhibitor at 15°C. Embryo is looking quite good and stable.


    Embryo is still lokking good and healthy (?). Letting it on… Stopped and saved the embryo. It moved out of the field of view around 1700.

  • Bruno Vellutini 16:22 on 2014/09/17 Permalink
    Tags: , mapk,   

    Membranipora temporary MEK inhibitor 

    Trying to understand better the effect of U0126 on Membranipora development I am starting a treatment with long pulses of the inhibitor first. Inhibitor will be washed every 8h.

    DMSO control 10 µM U0126
    10 µM U0126 washed after 9.5h 10 µM U0126 washed after 19h


    Interestingly, constant concentration shows reduced phenotype as usual, but the sample that was washed after 9h shows a higher number of relatively control like embryos. This might indicate that the effect of the MAPK inhibitor is either reversible, or, most probably, or unrelated to the observed phenotype which might be only a toxic general effect of the drug.

    Fixed one plate at 24h. The other for 48h.


    Same as previous sample. 9.5 hpa showed a higher proportion of normal larva then the 19hpa or the constant treatment. Fixwed with 4 % FA at RT for 1h.

  • Bruno Vellutini 16:15 on 2014/09/17 Permalink
    Tags: , , mapk, ,   

    Membranipora 4D U0126 10 µM 

    Started a recording BCV_Mm_2014_09_17 (old Mmem1_17_09_2014) with Membranipora in 10 µM U0126 MEK inhibitor at 15°C. Embryo is looking quite good and stable.


    Well, all the embryos were dead. Cells exploded and all. I think this was due to the high temperature of the room which made the slide temperature not be 15 °C as the ring should be, but more, killing the embryos.

  • Bruno Vellutini 21:02 on 2014/07/15 Permalink
    Tags: , mapk,   

    Anti-MAPK immuno staining Membranipora 

    I realized that I don’t have a proper description of all Membranipora stages with the anti-mapk antibody. I prepared 4 wells with mixed stages as following:

    S1: 3h, 10h, 11h, 13h, 16h S2: 18h, 22h, 26h, 30h, 42h
    S3: 46h, 50h, 68h, 70h, 88h S4: 3d, 4d, 5d, 9d
    1. Mix samples in PTx.
    2. Exchange 4x in 30 min.
    3. 2x 15 min PBT.
    4. 1x 40 min PTx+NGS.
    5. Incubate with Anti-MAPK 1:200 in 200 µL of PTx+NGS.


    1. 3x 5 min PBT.
    2. 4x 30 min PBT.
    3. 30 min PTx+NGS.
    4. 1:250 Anti-Mouse POD (Jackson) + Anti-Mouse HRP in PTx+NGS and incubate overnight in cold room at 21h.


    1. Washed out secondary with 3x 5min and 4x 30 min PBT.
    2. Washed 3x 10min in TNT buffer.
    3. Develop with TSA+Cy5 for 5 min.
    4. Stopped the reaction in PTx.
    5. 45 min with 1:4000 Sytox Green.
    6. Embedding with TDE.

    I realized that sytox staining was fading very quickly under fluorescent scope and decided to add 1:2000 sytox green into TDE and leave it overnight.


    Mounted and scanned sample 1 only in the confocal. They look nice, but maybe next time I would either stop the reaction with detergent solution or develop for shorter time, 3 minutes maybe.

    MAX_Mmem_MAPK_1.lif - Series003 MAX_Mmem_MAPK_1.lif - Series016 MAX_Mmem_MAPK_1.lif - Series022

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