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  • Bruno Vellutini 18:19 on 2013/11/20 Permalink
    Tags: , mapk, ,   

    Membranipora MAPK U0126 quantification 

    Before staining for the phenotype description I counted the number of normal embryos in the fine picked samples. Normal means late gastrula with apical lobe and elongated body axis.

    Seawater DMSO 1µM 10µM 25µM
    Normal late gastrula 31 31 33 0 0
    Weird 6 11 10 40 23
    Total 37 42 43 40 23

     
  • Bruno Vellutini 17:44 on 2013/09/24 Permalink
    Tags: , mapk, ,   

    MAPK inhibitor early stages 

    Used a 6-well plate with 10 µM U0126 to fix early embryos for immuno staining. Spawning activated at 12h and incubated at 14 °C.

    Embryos fixed after 8h.

     
  • Bruno Vellutini 15:43 on 2013/09/14 Permalink
    Tags: mapk,   

    Membranipora MAPK immuno staining 

    Use 10, 11, 13 and 16h samples. Do control without primary antibody (anti-MAPK) to see if staining is background from Cy5 of TSA kit.

    Control Anti-MAPK 1:200

    16/09/13

    Washed antibody off with 3x 5 min PBT and 5x 30 min PBT.

    17/09/13

    • Develop with TSA kit 1 µL fluorochrome + 49 µL of amplification reagent in 2 mL tube for 5 min.
    • Transferred embryos back to 4 well plate and washed 4x 5 min in PTx.
    • Selected a few embryos for staining: control dapi/phall, mapk dapi phall, and mapk sytox.
    • TSA treated embryos were stored in PBS at 4 °C.
    • Incubated with phallacidin for 2h, DAPI added 30 min before end. Sytox added 15 min before end.
    • Washed everything 3x with PBS.
    • Went through with gradient of Thiodiethanol (10, 25, 50, and 97%).
    • Mounted slides in TDE.
    • DAPI is horrible, phallacidin I don’t know, sytox is good.

    18/09/13

    Morning confocal time.

    Slides with TDE

    I don’t see any signal of the MAPK!! Is it gone with TDE??

    MAX_Mmem MAPK in TDE.lif - Series007 sytox MAX_Mmem MAPK in TDE.lif - Series023 phall

    Slides in glycerol

    No signal. I forgot to put the secondary!!!

    Added the secondary anti-mouse-pod…

    19/09/13

    Washed the secondary and developed with Cy5. Also stained with Sytox Green. Samples in PBS ready for TDE mounting.

    22/09/13

    • TDE steps: 10, 25, 50, 97% (3x).
    • Mounted slides, staining works.

    23/09/13

    Confocal time. Got some good pictures. There is extra background noise due to the extra TSA step… However, I think I could delimit the beginning and ending of MAPK activation. Also observed one embryo with 2 activated cells.

    PICS

     
  • Bruno Vellutini 17:33 on 2013/09/03 Permalink
    Tags: , mapk,   

    MAPK Membranipora immuno 

    Incubated Membranipora 10h embryos with MAPK antibody. Will leave it for 2-3d.

    06/09/13

    Washes with PBT and added Anti-Mouse POD (Jackson) + Anti-Mouse HRP 1:250 each. Incubated in the cold room.

    07/09/13

    Selected a few embryos to do the TSA amplification step only.

    1. Washed out secondary with PBT (3x5min and 4x30min).
    2. Transferred embryos to a 0.5 mL eppendorf and removed most of the liquid.
    3. Diluted DNP stock solution 1:50 in 1x amplification buffer from the kit and added to the tube for 5 min.
    4. Stopped the reaction with the Detergent Solution 2x 5 min at RT.
    5. Inactivated POD just to be sure incubating embryos in 0.1% H2O2 in PTx for 30 min at RT.
    6. Blocked 40min with 5% NGS in PTx.
    7. Incubate with Anti-DNP-HRP overnight 1:100 in 5% NGS at 4 °C.

    08/09/13

    • Wash with PBT 4x 15 min.
    • Wash with PTx 4x 30 min.

    09/09/13

    • TSA amplification step 1 µL of Cy5 + 49 µL amplification reagent.
    • Stop with 60 °C detergent solution + 1x detergent solution at RT.
    • Wash 3x with PTx.
    • Incubate with 1.25U of Phallacidin for 2h. After 1h added DAPI dilution to final volume of 1:1000 (250 Phall. + 50 µL of DAPI).
    • Washed samples 3x with PBS.
    • Confocal time!

    Detergent washes

    Apparently it does not make much difference of doing the detergent wash at 60°C or just PTx washes. Maybe the detergent hurt the phallacidin?

    DNP+TSA

    Doing the DNP amplification step before the TSA results in a lot of signal in the egg shell, which is not that good for imaging.

    TSA only

    Only the TSA seems to be enough for a stable signal and not so much background.

     
  • Bruno Vellutini 20:28 on 2013/08/21 Permalink
    Tags: , , mapk, ,   

    4D Membranipora with 10 µM U0126 

    Folder: BCV_Mm_2013_08_21

    Set an animal view of the embryo at 15 °C with 10 µM U0126. There was some weird blebbing in the beginning, so I thought it was over.

    22/08/13

    Looking ok, disruption in the 8 to 16 cell stage. Let it go.

    23/08/13

    Sabrina said it was fine.

    24/08/13

    Looking ok, still some cellular movements so letting it go.

    25/08/13

    ?

    26/08/13

    Someone turn the cooler and recording off around 15h. I rescued and fixed the embryo at 19h.

    Deleted from workstation stack X3001 onwards.

     
  • Bruno Vellutini 17:29 on 2013/08/20 Permalink
    Tags: , mapk,   

    10 µM U0126 for in situ, second try 

    Since the first try failed, I’m repeating it. Large batch of embryos put into 10 µM in 3 mL of FSW 3h post activation.

    22/08/13

    Fixing embryos after 33h for in situ.

     
  • Bruno Vellutini 16:07 on 2013/08/19 Permalink
    Tags: , mapk,   

    Second try MAPK immuno in Membranipora 

    Since the first try failed I’m increasing the antibody amount to 1:200 and doing a fluorescent A647 and a DAB/HRP development. Also, I picked embryos from the right stage, between 8 and 64 cells.

    AntiMAPK 1:200 + ALEXA647 1:250 AntiMAPK 1:200 + HRP/POD (2x) 1:250

    20/08/13

    Added secondary and let 2h at room temperature and put at 4 °C in the fridge (cold room is warm…).

    22/08/13

    Did the DAB-HRP reaction according to the wiki for 3 minutes. A lot of precipitate appeared and the solution became quite brown. I washed twice with distilled water and 3 times with 1x PBS. A few embryos were picked and transferred to a new well with phallacidin solution in PTx. Stained for 1h30 and added DAPI 1:1000 for 15 min and immediately transferred embryos to a slide with a drop of 80% glycerol in PBS.

    DAB-HRP

    DAB works well and it is visible. The only issue is that the egg membrane accumulates the precipitate and gets darker. I can think of something to solve this.

    Snap-7936

    DAB-HRP reaction works well and is quite visible.

    AVG_Stack

    Tried merge with DIC, Phallacidin, and DAPI…

    Composite

    Just a composite of Phallacidin and DAPI to show that the cytoplasm is much darker in the MAPK positive cell.

    Snap-7933

    The fluorescent antibody is very weak… with 2 slices scanned the signal was gone. And I had to push the laser up to get something. You can see from the pictures without and with a bit of the phallacidin channel. There is also some signal in other cells which does not appear with DAB.

    I still can do better with some different strategies in the confocal, but is there any way to amplify this signal? I didn’t try dab in the confocal, maybe some weird reflection works?

    Mm MAPK.lif - Series012 - s1

    Alexa 647 and DAPI channels. The real signal is super weak in the bottom right micromere. The rest I believe is background because I don’t see it in the DAB embryos.

     
  • Bruno Vellutini 18:14 on 2013/08/15 Permalink
    Tags: , mapk, ,   

    10 µM U0126 for in situ 

    Set a large batch of U0126 treated embryos to fix for in situ in 5 mL.

    17/08/13

    Many embryos disrupted and many with radialized phenotyped.

    18/08/13

    I was going to fix today, but they are all dead… need to repeat it.

     
  • Bruno Vellutini 08:30 on 2013/08/14 Permalink
    Tags: , mapk, ,   

    Membranipora inhibitor time points 14-24h 

    Adding more time points, from 14-24h post activation every 2 hours.

    CONTROLDMSO 1:500A: 18h30 U0126 10 µM1:1000A: 18h30
    I: 8h30 (14h)  
    I: 10h30 (16h)  
    I: 12h30 (18h)  
    I: 14h30 (20h)  
    I: 16h30 (22h)  
    I: 18h30 (24h)  

    15/08/13

    It seems that the difference between control and treatment is that control larvae look slender while treated seem to be shorter and wider. Will fix tomorrow, after 3d post activation.

    16/08/13

    Fixed.

     
  • Bruno Vellutini 20:38 on 2013/08/13 Permalink
    Tags: , , mapk, ,   

    4D Membranipora with 10 µM U0126 15 °C 

    Folder: BCV_Mm_2013_08_13

    Recording went overnight at 15 °C. Embryo rescued and fixed at 15/08/13 (2d). Optics are not the best, but it is visible. Also extends way past the needed stage, I think, but keeping all the files for now.

     
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