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  • Bruno Vellutini 18:11 on 2013/10/13 Permalink
    Tags: confocal,   

    Comparing Murray Clear and TDE 

    I want to compare mounting with Murray Clear and TDE and decide what is the best option for analysing the morphology of Terebratalia. Murray seems to clear the yolk while TDE only optimizes the refraction index of the immersion liquid. For this I got Terebratalia late larvae and will stain them with phallacidin and propidium iodide.

    • Got Terebratalia late larvae (6 and 7 days).
    • 3x quick washes in 0.2% PTx.
    • 2x 5 min 0.2% PTx.
    • 2x 10 min PBT.
    • Incubated 2h using 1.25 U in 250 µL of Phallacidin (6.25 µL of stock) and 1:500 dilution of propidium iodide.
    • Mount in Murray Clear and TDE.
    • Check staining in the confocal.

    Murray Clear + Phall/PI

    MAX_Ttra TDE vs Murray - Phall-PI.lif - Series007 Murray-3

    Murray Clear embryos shrank a lot and the morphology looks quite altered. Other than that, embryos are clearer and phallacidin works.

    TDE + Phall/PI

    MAX_Ttra TDE vs Murray - Phall-PI.lif - Series015 TDE-3

    TDE is imcompatible with phallacidin and we get a artefact general cytoplasm staining. However, the morphology is quite preserved and you can scan the bottom regions of the sample well.

  • Bruno Vellutini 18:51 on 2013/10/11 Permalink
    Tags: confocal, ,   

    Terebratalia engrailed immuno staining 

    I will try using the engrailed/invected 4D9 antibody in Terebratalia.

    • Get radial, asym., bilateral, and trilobed larvae (used mixed stages from Kristineberg samples).
    • Permeabilize with 0.2% PTx (5x 5 min, 1x 30 min).
    • 2x 15 min PBT.
    • 1x 30 min 5% NGS.
    • 17h45: incubate with primary on a tube with 100 µL and dilution of 1:20.


    • Wash out primary antibody along the day (3x 5 min PBT, 4x 30 min PBT, 1x 30 min PTx+NGS).
    • Incubate with secondary anti-mouse-pod 1:250 overnight.


    • Washed 3x 5 min in PBT.
    • 4x 30 min PBT.
    • Develop with TSA and Cy5 fluorochrome (49 µL diluent + 1 µL fluorochrome).
    • Stop the reaction with 1x detergent solution at 60 °C and 2x at RT.
    • Washed 3x 5 min in 0.2% PTx.
    • Stained with phallacidin for 2h.
    • Washed 3x 5 min in PBS.
    • Grading series with TDE and mounted in 97% TDE with DAPI.


    Confocal time. Engrailed antibody is indeed not working. I could try a higher concentration, maybe..

    Ttra DAPI-Phall TDE 1.lif - Series018 Ttra DAPI-Phall TDE 1.lif - Series021 Ttra DAPI-Phall TDE 1.lif - Series024 Ttra DAPI-Phall TDE 1.lif - Series027

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

    MAPK Membranipora immuno 

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


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


    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.


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


    • 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?


    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 16:07 on 2013/08/19 Permalink
    Tags: confocal, ,   

    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


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


    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 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.


    DAB-HRP reaction works well and is quite visible.


    Tried merge with DIC, Phallacidin, and DAPI…


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


    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 19:34 on 2013/08/13 Permalink
    Tags: confocal, ,   

    Membranipora anti-MAPK 3h, 22h, 26h, 50h, 70h immunostaining 

    Started immuno staining with freshly fixed material of Membranipora for 3h, 22h, 26h, 50h, 70h post fertilization.

    Anti-MAPK 1:1000 incubated at 19h30.


    Did regular washes and added secondary antibody Alexa 647 1:250.


    Washed in PBT and let at 4 °C in the cold room.


    Stained with phallacidin and DAPI and prepared a slide in glycerol. Both seem to be working fine, but I couldn’t see the MAPK signal under the fluorescence scope. So I need to see it under the confocal.


    Checked under the confocal and they were not good enough. Phallacidin was fading quickly, DAPI was weak and there was no clear signal for the MAPK. I’ll try the original concentration and DAB next time.

    MAX_Mmem DAPI PHALL MAPK A647.lif - Series014 MAX_Mmem DAPI PHALL MAPK A647.lif - Series018 MAX_Mmem DAPI PHALL MAPK A647.lif - Series044

  • Bruno Vellutini 18:31 on 2013/03/22 Permalink
    Tags: confocal, , , ,   

    More confocal of en+wnt doubles 

    Took some lateral views of en+wnt1 and a few scans of en+wnt5. The latter is quite noisy for wnt5 probe unfortunately…

    MAX_Ttra_en+wnt5.lif - Series000

  • Bruno Vellutini 19:27 on 2013/03/07 Permalink
    Tags: confocal, , , ,   

    Fluorescent in situ of Terebratalia engrailed 

    Mounted a slide with Terebratalia embryos from the first fluorescent/double in situ I tried for the segmentation genes and observed under the confocal. Mounting media had DAPI (magenta) and en is green. Although there is background it is possible to distinguish the real signal.

    Here are some max-projections to illustrate:

    MAX_Ttra En_DNP + DAPI.lif - Series031 MAX_Ttra En_DNP + DAPI.lif - Series028 MAX_Ttra En_DNP + DAPI.lif - Series031

  • Bruno Vellutini 20:03 on 2012/05/24 Permalink
    Tags: confocal, , , ,   

    Confocal of Vasa and Nanos fluorescent in situ 

    Acquired some stacks from the first fluorescent in situ in Terebratalia.

    Vasa: in early and late gastrula it is possible to see a stronger signal in the expected region (where the regular in situ shows expression), but there is still a lot of background noise masking the signal.
    Nanos: signal is even weaker and I could not identify it in the gastrula stage. Later stages need a better positioned larva, but the scanned sample shows a faint signal where the expression should be.

  • Bruno Vellutini 15:30 on 2012/05/24 Permalink
    Tags: confocal,   

    Confocal imaging of Membranipora 

    In order to get a better characterization of the cyphonautes larvae I am staining with Phallacidin and Propidium Iodide (1:500). Just checked the slide under confocal and the staining looks good.

  • Bruno Vellutini 13:00 on 2012/05/01 Permalink
    Tags: confocal,   

    Captured confocal images of the L. ruber fixed with and without cysteine treatment in samples not treated with Murray’s clear. Stained with Phallacidin and Propidium Iodide. It seems that the juvenile tissues are well preserved and with no disruptions, although I could see few epithelial cells that apparently popped off.

    Using the 20x objective I could acquire whole body images and also a closeup of the anterior end in 2 individuals. Early stages (10d) seem to be only rubbish, strange blastomeres whose nuclei were not stained. Seem to be degenerating. Probably unfertilized eggs or interrupted embryos, although I still need to analyze the images properly.

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