Tagged: Membranipora membranacea Toggle Comment Threads | Keyboard Shortcuts

  • Bruno Vellutini 16:37 on 2015/08/25 Permalink
    Tags: , Membranipora membranacea   

    Membranipora simple staining 

    Just a simple staining of mixed stages of Membranipora using DAPI and Phalloidin 647 for morphology.

    • 3x 30min 0.2% PTx washes.
    • 1x PTx overnight at 4°C.

    26/08/15

    • Staining with Phalloidin and DAPI for 2h.
    • Let embryos in PBS+DAPI 1:1000 at 4°C.

    It failed… weird.

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

    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.

    26/08/15

    • 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.

    27/08/15

    • 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, , Membranipora membranacea, 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
    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 22:57 on 2015/05/11 Permalink
    Tags: Membranipora membranacea, , , ,   

    Long probe for Novocrania wnt1 

    Set probe PCR for:

    Na wnt1 BV387: T7 80 ng/µL
    Mm pl10 BV79: SP6 702 ng/µL

    and extracted.

    12/05/15

    Set transcription reaction and precipitation.

    13/05/15

    Finished probes (see values above) and made 1x dilution for wnt1.

     
  • Bruno Vellutini 18:24 on 2015/05/08 Permalink
    Tags: , , , , , Membranipora membranacea, , , , , ,   

    Membranipora in situ and Novocrania wnt1 

    Starting in situ in Membranipora with dorsoventral genes and additional genes that we backgroundish.

    wnt1 dlx bmp2/4 chordin fgf
    nanos vasa piwi1 piwi2 pl10
    Na wnt1

    Standard in situ protocol with 10 min protk. Washes after glycine were slightly shorter and fixing went for 1:30h. Used 83°C for fosfatase step and prehybe at 67°C.

    09/05/15

    Added probes just after synthesis.

    11/05/15

    Novocrania were dissolved. The rest were fine. Continued with hybe washes and added anti-dig-ap.

    12/05/15

    Antibody washes. Plus developing. dlx and piwi1 were stopped after a couple of hours. The remaining were developed overnight at 4 °C.

    13/05/15

    Exchanged the AP and stopped the reaction around 1200. Did ethanol washes and left in 70% glycerol + dapi overnight at room temp.

     
  • Bruno Vellutini 18:29 on 2015/04/29 Permalink
    Tags: , , , , Membranipora membranacea, , ,   

    Membranipora cloning final genes 

    Set PCR with:

    Mm bmp2/4 Mm otx1 Mm chordin Mm fgf8/17/18
    Mm nanos Mm piwi1 Mm piwi2 Mm vasa

    30/04/15

    Ran gel:

    2015-05-01 16.23.27

    Most important genes worked: bmp2/4, chordin and fgf8. piwi1 is bonus. Extract these and set a ligation for 3h. Transformed and plated around 18h.

    01/05/15

    Set colony PCR.

    2015-05-01 16.23.36

    Put colonies to grow.

    02/05/15

    Extracted minipreps, set sequencing PCR and dropped at seq facility.

    BV378 Membranipora membranacea Bmp2/4 T7 ok + SP6
    BV379 Membranipora membranacea Bmp2/4 T7 ok + SP6
    BV380 Membranipora membranacea Chordin T7 ok + SP6
    BV381 Membranipora membranacea Chordin T7 ok + SP6
    BV382 Membranipora membranacea Fgf8/17/18 T7 ok + SP6
    BV383 Membranipora membranacea Fgf8/17/18 T7 ok + SP6
    BV384 Membranipora membranacea Piwi1 T7 ok + SP6
    BV385 Membranipora membranacea Piwi1 T7 ok + SP6

     

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

    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 09:50 on 2015/04/05 Permalink
    Tags: , , , , , , , , Membranipora membranacea, , , , ,   

    Membranipora in situ with mesodermal genes 

    First in situ with mesodermal genes of Membranipora plus some extras. Will add a control with Chema’s probe and do only early stages until late gastrula so I don’t have to worry with cyphonautes larvae.

    foxa foxc foxd foxf
    mef2 noggin2 eya (long) mprx
    fhl mox pax6 wnt4

    ProtK lasted 10min30s but washed with 800/500 µL. Otherwise standard protocol. They stayed longer (30min) in the first hybe buffer until the oven was free. Pre-hybe in the oven at 67 °C.

    06/04/15

    Put probes.

    08/04/15

    Standard washes as usual. Stayed 1h30 in 1x blocking before I added the antibodies.

    09/04/15

    Wash off secondary antibody. Started developing. Most genes did not show any clear staining except for pax6 in mid gastrula and foxa. Background was much better than the previous in situ, but not completely absent. Maybe try an even higher temperature?

    10/04/15

    Now there is visible signal in all fox genes: early single cell expression in early gastrula and in the anterior muscles of the late gastrula. eya expression also came up in some internal cells. Other genes have no clear signal. I stopped all the reactions by the end of the day.

    11/04/15

    Ethanol washes and placed in 70% glycerol with DAPI.

    12/04/15

    I’ll mount some slides and check the patterns.

     
  • Bruno Vellutini 18:45 on 2015/03/30 Permalink
    Tags: , , , Membranipora membranacea, , , , ,   

    Membranipora wnt in situ 

    First new try with bryozoan in situ. Separate wells for early (until extended gastrula) and late stages (early cyphonautes and later cyphonautes).

    early stages 1x nk2.1 1x wnt1 1x wnt5 1x wnt7 1x wnt8
    late stages 0.1x nk2.1 0.1x wnt1 0.1x wnt5 0.1x wnt7 0.1x wnt8
    early stages 1x dlx 1x gbx 1x pax258 1x nanos 1x vasa
    late stages 0.1x dlx 0.1x gbx 0.1x pax258 0.1x nanos 0.1x vasa

    After protk step (10 min) embryos were suspended in glycine (added 800 µL) and did not sink. I added 600 µL of glycine for the second wash. The whole glycine steps lasted for 15 min. The remaining washes were a bit longer than 5 min, but I could control it better.

    I noticed that the embryonic membrane dissolves with protk.

    Let overnight in pre hybe.

    31/03/2015

    Diluted and added probes to in situ (first 0.1x then 1x). Left oven set to 63 °C (62 in the electronic thermometer and 63 in the mercury thermometer).

    02/04/2015

    First hybe wash I removed 1x probes first, filled with warm hybe wash and then removed 0.1x probes and added warm hybe.

    • 1x 10min hybe wash
    • 1x 50min hybe wash
    • 1x 30min 75% hybe
    • 1x 30min 50% hybe
    • 1x 30min 25% hybe
    • 1x 30min 2x SSC
    • 3x20min 0.2x SSC
    • 1x 10min 75% 0.2x SSC
    • 1x 10min 50% 0.2x SSC
    • 1x 10min 25% 0.2x SSC
    • 1x 10min PTw.
    • 4×10-15min washes in PBT (total 45min)
    • 1h in 1x blocking buffer
    • Overnight at 4°C with anti-dig-ap

    03/04/2015

    • 3x 5min PBT
    • 4x 15min PBT
    • 5x 30min PTw

    Developed in situ. A significant amount of pinkish background came up in my probes. wnt5 was fast, also dlx. Others had different levels of background in addition to the real signal, except Chema’s probe for NK2.1. Thus, this background is likely due to probe design or probe synthesis. Here are the lengths of the clones used to synthesize the probes:

    gene nk2.1 wnt1 wnt5 wnt7 wnt8 dlx gbx pax2/5/8 nanos vasa
    bp 1113 1038 1073 869 886 823 888 902 868 812

    Length of the probe can be the issue but it does not quite explain the rapid background coming up. Longer probes should result in a better signal to noise ratio because the signal comes up faster and not because the background comes up slower. It can also be something related to the probe synthesis process. But what? I usually get high yields and good peak ratios. Check the numbers to see!

    Ah, one obvious thing is the probe concentration. NK2.1 probe is probably more diluted than the others since it is its second or third use. Thus, the next in situ should be lower concentration or higher temperature.

    04/04/15

    Continued developing wnt7 and gbx and stopped them after 3h. Ethanol washes with 20min in 100% and let in PTw overnight at 4° C because glycerol was not ready.

    05/04/15

    Putting them in 70% glycerol with DAPI. Will put apart some of the NK2.1 embryos to do a MAPK immuno staining.

     
  • Bruno Vellutini 18:39 on 2015/03/23 Permalink
    Tags: Membranipora membranacea   

    Cloning Membranipora mesodermal genes 

    Cloning mesodermal genes in Membranipora: wnt4, pax6, noggin, mox, paraxis, foxf, mprx, nog2, mef2, fhl, foxc, foxd, eya. Nano wnt1 longer probe 2try.

    24/03/15

    2015-03-23 19.57.29

    25/03/15

    2015-03-25 16.45.11

    26/03/15

    Extract minipreps and sequencing. Done. BV359 to BV377 with T7 primer.

    BV359 Membranipora membranacea FoxC T7 ok + SP6
    BV360 Membranipora membranacea FoxC T7 ok + SP6
    BV361 Membranipora membranacea FoxD T7 ok + SP6
    BV362 Membranipora membranacea FoxF T7 ok + SP6
    BV363 Membranipora membranacea Mef2 T7 ok + SP6
    BV364 Membranipora membranacea Mef2 T7 ok + SP6
    BV365 Membranipora membranacea Noggin2 T7 ok + SP6
    BV366 Membranipora membranacea Noggin2 T7 ok T7
    BV367 Membranipora membranacea Eya T7 ok + SP6 short isoform
    BV368 Membranipora membranacea Eya T7 ok + SP6 long isoform
    BV369 Membranipora membranacea Eya T7 ok T7
    BV370 Membranipora membranacea mPRX T7 ok + SP6
    BV371 Membranipora membranacea mPRX T7 ok T7
    BV372 Membranipora membranacea FHL T7 ok + SP6
    BV373 Membranipora membranacea Pax6 T7 ok T7
    BV374 Membranipora membranacea Pax6 T7 ok + SP6
    BV375 Membranipora membranacea Mox T7 ok T7
    BV376 Membranipora membranacea Wnt4 T7 ok + SP6
    BV377 Membranipora membranacea Wnt4 T7 ok + SP6

     
c
Compose new post
j
Next post/Next comment
k
Previous post/Previous comment
r
Reply
e
Edit
o
Show/Hide comments
t
Go to top
l
Go to login
h
Show/Hide help
shift + esc
Cancel