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  • Bruno Vellutini 17:48 on 2012/05/31 Permalink
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    Membranipora colonies are alive and ok. Most of them do not have gametes still, though spermatozeugma are present. Changed more than half of the water of the buckets.

     
  • Bruno Vellutini 09:41 on 2012/05/30 Permalink
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    Went collecting Membranipora with Anette at Hjellestad:


    View Larger Map

    The dock has a lot of kelp growing and the colonies of Membranipora and Electra were larger than last time, but still small (around 5 cm). We could find large colonies covering the whole surface of the algae. We also collected a jellyfish for muscle staining.

    Leaves were distributed in half-full buckets with water at 4 °C. I checked the colonies and most of the large ones had plenty of sperm inside the zooids, but not disc shaped eggs. There were smaller and rounded oocytes apparently. Buckets kept with a black plastic bag in the cold room.

    30/05/12

    Checked some colonies to see if they were alive; they were. Removed 1/3 of the water from the buckets and added more clean sea water.

     
  • Bruno Vellutini 18:00 on 2012/05/29 Permalink
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    We collected a jellyfish for muscle Phallacidin staining for Andi’s News & Views article. I cut small pieces of the marginal region of the umbrella and followed the standard fixation protocol for confocal stainings. I also cut larger portions of the same region and fixed in 4% PFA overnight at 4 °C.

    30/05/12

    Washed the larger portions in PBS+Triton-X during the whole day and transferred to PBS at 4 °C. I stained one well with Phallacidin and looked in the fluorescent lamp to find the muscle layer. It is an opaque sheath of tissue below the mesoglea. The staining worked.

    31/05/12

    Dissected the muscle layer better and mounted the slide. The tissue is well spread and staining looks ok. Confocal tomorrow.

    01/06/12

    Confocal time.

     
  • Bruno Vellutini 18:00 on 2012/05/26 Permalink
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    Took pictures of Terebratalia in situ for germline genes vasa, nanos, piwib.

     
  • Bruno Vellutini 15:22 on 2012/05/25 Permalink
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    In situ segmentation genes Terebratalia 

    Starting an in situ for segmentation genes in Terebratalia and repeating the fluorescent in situ for Vasa and Nanos, trying to reduce the background. The plate looks like this:

    netrin patched hedgehog vasa
    smo2 nk2.2 nk5 nanos

    For vasa and nanos I will use a lower concentration of the antibody, 1:500 (last time it was 1:100) — effort to reduce background. For nanos I will also double the amount of probe to get a stronger signal.

    Samples put in prehybe at 17h:00.

    26/05/12

    Added regular probes at 1 ng/µL, except for nanos which was 2 ng/µL (trying to increase the signal/noise ratio for fluorescent in situ).

    28/05/12

    Washes. Added regular Anti-Dig/AP to segmentation genes (1:5000) and Anti-Dig/POD (1:250) to vasa and nanos (in an attempt to lower the background noise — used 1:100 previously).

    29/05/12

    Regular washes with PBT and PTw.

    30/05/12

    Started developing segmentation genes of Terebratalia at 11:30. For the fluorescent in situ I developed normally for 1h30 and then stopped the reaction with a detergent solution used by (10.1038/nature10838) to reduce the background; 3 washes of 20 min at 62 °C. The solution:

    [list of chemicals]

    I checked the slides in the fluorescent lamp and did not see any staining. It is also not so clear if there is less background. I guess I need to check in the confocal.

    The segmentation genes began to appear. Netrin is the strongest one and the pattern is interesting. I notice that the “ring” background might be covering the whole anterior of the pedicle lobe. Other genes are still weak to say something.

    Changed the AP substrate at 16:30 and let it developing at 4 °C.

    31/05/12

    Patterns are more clear today. Changed the substrate 3 times (10:15, 12:30, 16:00) and let it overnight at 4 °C. Crystal began to appear after lunch and are already covering the whole bottom of the well.

    01/06 – 04/06

    Stopped netrin, nk2.2, and nk5. Developed others until 04/06. Many crystals forming…

    Bibliography

     
  • Bruno Vellutini 20:03 on 2012/05/24 Permalink
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    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
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    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 15:19 on 2012/05/24 Permalink
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    Primers for more Terebratalia germline genes have arrived.

    • Tt dicer1b
    • Tt runx2
    • Tt maelstrom
    • Tt tudor1
    • Tt pl10
    • Tt ago
    • Tt pum
     
  • Bruno Vellutini 17:20 on 2012/05/22 Permalink
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    Fed adult nemerteans. Noticed that I should chop the shrimp pieces really small, otherwise they give up swallowing and abandon the food. It seems that they lose interest quite fast (~15 min), so maybe it is better to clean the water instead of leaving the food overnight.

    Tried to feed 2 months old juveniles, but they were not interested in the shrimp piece.

     
  • Bruno Vellutini 12:03 on 2012/05/18 Permalink
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    Terebratalia in situ for germline genes (+Membranipora testing) 

    First in situ using segmentation genes for Terebratalia transversa. The list is:

    Tt netrin BV110 SP6
    Tt patched BV115 SP6
    Tt hedgehog BV129 SP6
    Tt smoothened2 BV131 SP6
    Tt nk2.2 BV135 SP6
    Tt nk5 BV139 SP6
    Mm piwi1 BV94 SP6
    Mm gata123

    Put in prehybe at 19:45.

    19/05/12

    Probes for Terebratalia genes were no good, so changing to Vasa, Nanos and Piwi:

    Tt vasa
    Tt nanos
    Tt piwi
    Tt vasa (fluo)
    Tt nanos (fluo)
    Tt piwi (fluo)
    Mm piwi1
    Mm gata

    Put the probes and let hybridizing at hybe temp.

    21/05/12

    First day of washes. Added Anti-Dig/POD (1:100) to the 3 wells of vasa, nanos, and piwi and the regular Anti-Dig/AP for the remaining; 19h and let overnight at 4° C.

    22/05/12

    Washing and started developing the regular in situ in 5 wells overnight at 4 °C.

    23/05/12

    Changes in AP Substrate: 10:30, 12:00, 14:30

    AP is becoming purpleish quite rapidly; patterns are developing accordingly fast and as expected for Terebratalia genes. Membranipora Piwi1 shows no sign of staining (although it seems to be a bit pinkish) and GATA123 is appearing, although it is not easy to identify the pattern (or if it is similar to Andi’s in situ).

    At 11:00 I started washing and then developing the fluorescent in situ. Checking under the 4D room scope the embryos show staining, but it is hard to say if the in situ worked, yet. I need to look mount them and look in the scope.

    24/05/12

    Mounted fluorescent in situ samples after staining with DAPI (1:1000) and checked in the scope. Vasa is the strongest, but still a bit weak. So next time it is better to use more probe. Nanos is there, but barely visible and Piwi is difficult to say, since the expression is widespread. Made images in the confocal and the signal seems to be there, but there is a lot of background.

    Stopped Vasa regular in situ at 10:00 and changed the substrate for the others. Changed again at 13:30. Yellow crystals were present in the Membranipora wells. Changed at 16:30 and left overnight at 4 °C.

    25/05/12

    Stopped all wells and stored in 70% glycerol.

     
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