[Xenopus] high-throughput devitellinizing?
Richard Harland
harland at berkeley.edu
Sat Oct 31 13:29:50 EDT 2020
There are several protocols out there for helping remove the membrane. Depending on what you have handy.
I use proteinase K,, 5 microliters of 10 mg/ml stock in a small petri dish. Once the membrane starts to be visible and lift just a little, then of course it's all about rinsing and putting in competitor proteins (smashed embryos). The membrane continues to lift for a while. Overdigesting effects are obvious!
prot k
http://cshprotocols.cshlp.org.libproxy.berkeley.edu/content/2007/5/pdb.prot4732.long <http://cshprotocols.cshlp.org.libproxy.berkeley.edu/content/2007/5/pdb.prot4732.long>
trypsin
https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1440-169X.1972.00037.x <https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1440-169X.1972.00037.x>
or fixed embryos
https://www-sciencedirect-com.libproxy.berkeley.edu/science/article/pii/0168952596999904?via%3Dihub
> On Oct 31, 2020, at 9:03 AM, Levin, Michael <Michael.Levin at tufts.edu> wrote:
>
> Hi everyone,
>
> From: Enrique Amaya <Enrique.Amaya at manchester.ac.uk>
>> The most time consuming part is removing the vitelline membranes
>
> Yes!! Speaking of which: does anyone have an enzymatic method for this yet? Maybe recombinant hatching enzyme cocktail or something? Is there a decent way to devitellinize tons of embryos at once?
>
> Best,
>
> Mike Levin
>
>
> ---
> Dr. Michael Levin
> Vannevar Bush Chair,
> Biology Department, and
> Director, Allen Discovery Center at Tufts and
> Tufts Center for Regenerative and Developmental Biology
> Tufts University
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> -----Original Message-----
> From: <xenopus-bounces at lists.mbl.edu> on behalf of Enrique Amaya <Enrique.Amaya at manchester.ac.uk>
> Date: Thursday, October 29, 2020 at 7:45 AM
> To: Jeremy Green <jeremy.green at kcl.ac.uk>
> Cc: Leon Peshkin <peshkin at gmail.com>, "xenopus at lists.mbl.edu" <xenopus at lists.mbl.edu>
> Subject: Re: [Xenopus] MISC
>
> Dear Leon,
> The most time consuming part is removing the vitelline membranes. When I was animal cap king decades ago my preferred method was to use an eye brow hair knife and put the de-vitellinised blastula embryos on their side and decapitate them with the eye brow knife… a very rapid method. If working with another, I would suggest having someone removing the vitelline membranes and another person performing the decapitations, to ensure consistency in animal cap removals. One can easily get through hundreds this way. How many do you need?
> Enrique
>
> ——————————————————————————————
> Professor Enrique Amaya
> Division of Cell Matrix Biology & Regenerative Medicine
> Michael Smith Building
> School of Biological Sciences
> Faculty of Biology, Medicine and Health
> University of Manchester
> Oxford Road
> Manchester M13 9PT
> United Kingdom
> Office: +44 161 275 1716
> Twitter: @Enrique1Amaya
>
>
>
>
> On 29 Oct 2020, at 11:08, Green, Jeremy <jeremy.green at kcl.ac.uk> wrote:
>
> Dear Leon,
> With a bit of practice and well-sharpened Dumont #5 forceps, a person can cut around 200 caps in an hour. I suggest that if you want to get many hundreds of caps that you simply have a cap-cutting jamboree - not so much biting the bullet as having a party. Seriously. I think you’ll have no problem getting volunteers and (COVID, etc. permitting), I’d be happy to come over myself.
>
> Best wishes,
>
> Jeremy
>
> Jeremy B.A. Green Ph.D.
> Vice-Dean International (Research)
> Professor of Developmental Biology
> Centre for Craniofacial & Regenerative Biology
> King's College London
> Guy's Tower, Floor 27
> London SE1 9RT
> UK
>
> jeremy.green at kcl.ac.uk
> Tel. +44 20 7188 1794
> https://www.kcl.ac.uk/people/jeremy-green
>
>
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> On 29 Oct 2020, at 03:45, Leon Peshkin <peshkin at gmail.com> wrote:
>
> Dear Colleagues !
>
> I have a few general questions and tried posting to Slack "Xenopus Community" without success. People - Slack is a nice way to keep discussions going, please use it !
>
>
> Has someone worked out a way to make animal caps en masse ? I did hear the gastromaster legend and even obtained one with a few tips … but it does not really work in my hands. Any other devices ? Please do not tell me to just bite the bullet. I'd like to be able to cut many hundreds.
>
> There are a couple of animal cap instructional videos with forceps but I know many people use other techniques. Would be nice to have more videos.
>
>
> Does someone have a protocol and experience for blood perfusion in Xenopus ?
>
>
> THANK YOU
>
>
> - L. Peshkin
>
>
>
>
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