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

    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: , , , , , , , vasa   

    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 18:45 on 2015/03/30 Permalink
    Tags: , , , , , , , vasa,   

    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 19:30 on 2012/11/16 Permalink
    Tags: brachyury, , , , , , , , vasa   

    Lineus ruber and Membranipora in situ 

    Starting the first in situ in the nemertean Lineus ruber and trying different genes in Membranipora after establishing the ProtK timing. Selected genes are:

    L. ruber foxa six3/6 cdx gsc
    M. membranacea bra gsc piwi1 vasa

    All wells treated with 10 min in 1x protk concentration (0,01 mg/mL).

    17/11/12

    Added probes.

    19 and 20/11/12

    Washing days, normal activity. Samples stayed in blocking buffer for 1h30 (instead of 1h) due to epic foosball match.

    21/11/12

    Began developing at 11h. Twenty minutes later Mm bra cyphonautes shell were already completely blue… It seems that mucus cells in Lineus juveniles are being stained. No real signal coming up, except in six3/6, maybe. Mm piwi1 and vasa look like to be working, something is appearing. Exchanged AP once and let it at 4°C.

    22/11/12

    Changed the AP of Lineus twice. Some signal kind of visible below the epidermis, nothing in the embryos. Stopped Membranipora development.

    23/11/12

    Patterns in Lineus more visible and apparently real signal. foxa expressed in the gut region, six3/6 in the brain/anterior, cdx in the posterior tip (not in the epidermis), and gsc in the anterior mouth region. Let developing one more day at 4°C.

    25/11/12

    Stopped reaction. Background began to come up, but I could check the expression more clearly. For example, foxa is detected in the mouth region and cdx in the anus. Now what needs to be done is remove the mucus cells with cystein before fixation.

    26/11/12

    Restart development of Lineus to see if the signal get stronger in the juveniles and something appears in the embryos.

    29/11/12

    Finally stopped the development. Will do ethanol washes tomorrow.

     
  • Bruno Vellutini 18:01 on 2012/06/06 Permalink
    Tags: , , vasa   

    FITC probe and washes for fluorescent in situ 

    Chema developed Tt vasa using FITC probe for regular and fluorescent in situ and also Tt nanos. He washed the fluorescent ones according to the protocol from (10.1186/2041-9139-2-6) (basically the 20 minutes wash with detergent), which consists of:

    Fixed larvae hybridized with Digoxigenin-11-UTP (Roche Applied Science, Indiana- polis, IN, USA) labelled Tt-c-opsin and Fluorescein-12-UTP (Roche Applied Science, Indianapolis, IN, USA) labelled Tt-Pax6 probes at 2.5 ng/μl each, for 48 hours at 60°C. Larvae were incubated overnight with Anti-digoxigenin-POD antibody (Roche Applied Science, Indianapolis, IN, USA) at a 1:1000 dilution in 1× Blocking Reagent (Roche Applied Science, Indianapolis, IN, USA) overnight at 4°C, and stained with TSA Plus Cyanine 5 at a 1:100 dilution for 60 minutes at room temperature. Following staining, peroxidase activity was extinguished by incubation in 2.7% hydrogen peroxide in 1× PBS buffer for 90 minutes. Subsequently, larvae were incubated overnight with Anti-fluorescein-POD antibody (Roche Applied Science, Indianapolis, IN, USA) at a 1:1000 dilution in 1× Blocking Reagent (Roche Applied Science, Indianapolis, IN, USA) overnight at 4°C, and stained with TSA Plus Tetramethylrhodamine at a 1:100 dilution for 60 minutes at room temperature. Stained larvae were washed for 48 hours with multiple exchanges of 1× PBS buffer, and cleared with 80% glycerol.

    The washes apparently reduced the background, though also the signal. I’ll check the samples in the confocal.

    Bibliography

     
  • Bruno Vellutini 18:00 on 2012/05/26 Permalink
    Tags: , , , , , , vasa   

    Took pictures of Terebratalia in situ for germline genes vasa, nanos, piwib.

     
  • Bruno Vellutini 15:22 on 2012/05/25 Permalink
    Tags: , , , , , , , , vasa   

    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
    Tags: , , , , vasa   

    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 13:40 on 2012/05/04 Permalink
    Tags: , , , , , vasa   

    In situ Mm Nanos and Tt Vasa, Nanos, Piwib 

    Trying 2 samples of late cyphonautes of Membranipora, one without proteinase-k treatment and one with 30s half-concentrated dose. Also, samples of all stages of Terebratalia using the probes for Vasa, Nanos, and Piwib.

    Mm Nanos Mm Nanos Tt Vasa Tt Nanos Tt Piwib
    no prot-k 30s half-conc prot-k 10min prot-k 10min prot-k 10min prot-k

    Membranipora embryos/larvae take 1 min to sink. When trying to do 30s of prot-k I managed to start removing the solution at 15s, but only finished and added glycine around 45s. I added a double dose of glycine to dilute the prot-k well and quickly did the second wash (also with double amount). Membranipora embryos treated with prot-k were fixed for 1h30 while Terebratalia for 1h.

    05/05/12

    Added the probes to the wells and let it hybridize over the weekend.

    07/05/12

    First day of washes. Membranipora embryos look ok (not dissolving) and Terebratalia became a bit more transparent, but also look integer. Put the antibody at 18:30, incubating overnight rocking at 4 °C.

    08/05/12

    Washes with PBT and PTw ran as expected. Letting overnight at 4 °C. Embryos are still there and apparently no differences between prot-k treatment and control of Membranipora.

    09/05/12

    Developing from 11h.

    30min: Apparently nothing on Membranipora and Terebratalia Nanos and Piwib. Terebratalia Vasa is already showing the expression, a bit more broadly expressed in gastrula stages and restricted to a ring in the mid-lobe of larvae.
    60min: Nothing on Mm. Tt Nanos and Piwib started to develop a faint staining in the archenteron region of the gastrula. Tt Vasa continues to show a strong staining pattern.
    90min: Same.
    3h: TtVasa looks stronger and TtNanos starting to appear more clearly.
    5h: Same pattern. Changing the AP substrate and leaving at 4 °C overnight.

    10/05/12

    Stopped Tt Vasa at 10h (following PTw washes). Changed the AP substrate for the others. Signal is stronger in Nanos and Piwib, better revealing the pattern. Apparently nothing is coming up in Mm, although I saw some unspecific bilateral staining appearing.

    Changed AP substrate at 10h, 14h, and 18h. Leaving overnight at 4°C.

    11/05/12

    Forgot to filter the AP substrate, so wells were full of yellow crystals in the bottom and covering the samples… :( I stopped the reaction and did the Ethanol washes for all wells. Crystals were mostly dissolved, but wells became dirty. After ethanol washes it was all good again. Stored in the fridge with 70% glycerol in PTw.

    14/05/12

    Mounted Membranipora samples with and without proteinase-k. Both look similar and show no expression of Nanos in the expected region. So it seems that the in situ did not work again. At least the embryos made it to the end of the protocol in one piece.

     
  • Bruno Vellutini 18:00 on 2012/04/30 Permalink
    Tags: , , , , , , vasa   

    Probe synthesis for Mm Vasa, Nanos, Piwi1, Piwi2 and Tt Vasa, Nanos, Piwib 

    Running a probe PCR for germ line genes of Membranipora (again) and Terebratalia (see table below). Setup is the same as a colony PCR, but doubling the amount (50µL) and using 3 µL of plasmid DNA. I extracted the bands and purified the DNA.

    Membranipora probe pcr for nanos, piwi2, vasa, and piwi1Terebratalia probe pcr for vasa, nanos, and piwib

    01/05/12

    This is the gel with the purified yield and the nanodrop measures.
    Purified dna from probe pcr of Mm and Tt germ genes

    02/05/12

    Transcription reaction initiated at 11h, tubes put in the cycler using incubate function at 37 °C.

    03/05/12

    Finished the probe synthesis and stored in hybe buffer diluted 50 ng/µL.

    Probe specs
    ID Gene Probe Kit Probe PCR ng/µL  Probe ng/µL
    BV3 MmNanos SP6 881.0 1283.41
    BV6 MmPiwi2 SP6 745.1 1149.77
    BV91 MmVasa SP6 586.9 477.97
    BV94 MmPiwi1 SP6 571.3 902.3
    BV96 TtVasa SP6 517.8 572.53
    BV100 TtNanos SP6 582.4 1158.87
    BV103 TtPiwib SP6 651.8 493.36

     
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