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Organotypic Slice Culture of E18 Rat Brains

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1Institute for Regeneration Medicine, University of California, San Francisco - UCSF

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    Summary

    Date Published: 7/11/2007, Issue 6; doi: 10.3791/235

    Cite this Article

    Elias, L., Kriegstein, A. Organotypic Slice Culture of E18 Rat Brains. J. Vis. Exp. (6), e235, doi:10.3791/235 (2007).

    Abstract

    Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice.

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    Comments

    25 Comments

    Is it possible to download this video for educational purposes? It would be used as part of a lecture on biotechniques. If so how do I download it?Many thanks,Dervla O'Malley, PhDLaboratory of NeurogastrŒnterologyUCC, Cork, Ireland.
    Reply

    Posted by: AnonymousApril 10, 2008, 12:18 PM

    Hi Dervla,Please contact me directly at nikita.bernstein@jove.com and we will make arrangements.
    Reply

    Posted by: AnonymousApril 10, 2008, 12:27 PM

    Dear Laura, thanks for the nice movie. I want to ask you whether you have any special modification of the protocol when you use mouse instead of rat embryos. Have you ever tried with E15 mouse embryos? Thank you. Kind regards,   Angelika Vogt University of Heidelberg, Germany
    Reply

    Posted by: AnonymousMay 28, 2008, 5:53 AM

    Dear Angelika,

    Our lab uses the exact same protocol for mice.

    Also, it isn’t really age specific it just becomes a bit harder to make the slices the earlier you go, but I routinely make E15 rat slices which is around E13 for mouse so you shouldn’t have any problem.

    I am also putting the ACSF and slice culture recipes as that may be useful to have.

    ACSF recipe:

    R1:
    NaCl 73.05g/L
    KCl 1.86g/L
    MgCl²-6H²O ².03g/L
    CaCl²-²H²O ².94g/L
    NaH²PO4-H²O 1.73g/L
     
    R²:
    NaHCO3 ²1g/L
     
    Filter R1 and R² and store at 4 degrees
     
    To make ACSF –
    100mL R1
    100mL R²
    800mL H²0
    4.5g glucose


    Slice culture recipe:
    ²5mL Hanks BBS
    66mL Basal Medium Eagle
    5mL Fetal bovine serum
    1mL Pen/Strep
    ²mL glucose (33% stock)
    1mL N²-supplement (invitrogen)



    Best, laura
    Reply

    Posted by: AnonymousMay 30, 2008, 12:34 AM

    Dear Laura,   thanks a lot for your help!   Best, Angelika
    Reply

    Posted by: AnonymousMay 30, 2008, 5:34 AM

    Laura,

    How can be E15 mouse be same as E13 mice, as I think mouse E13 will be smaller in size than E15 rats. Please can you explain are you referring to size or development of embryo.

    thanks

    Suneet Mehrotra
    Reply

    Posted by: AnonymousSeptember 22, 2011, 6:05 PM

    Hi Suneet,

    I think here Laura means that E13 in mouse is equivalent to E15 in rat in terms of development stage, not the size. So it may be harder to section but it won't be a big problem. I have done sectioning in mouse brain at E11.5.
    Good luck!
    Shanzheng
    KI, Stockholm,Sweden
    Reply

    Posted by: AnonymousMarch 16, 2012, 6:10 AM

    Dear Laura, thanks for sharing your experience of culturing brain slice. I have a question about the choice of agarose, which kind of agarose you used to embed brains and is there any special requirement for agarose? Thank you! Wen Zeng Sichuan University, China
    Reply

    Posted by: AnonymousJune 2, 2008, 11:59 PM

    Dear Wen Zeng, We use low melt agarose (Fisher Scientific, BP165-²5, gelling temperature ²5). best, laura
    Reply

    Posted by: AnonymousJune 6, 2008, 4:29 PM

    Dear Laura, Thanks for your help. Best, Wen Zeng
    Reply

    Posted by: AnonymousJune 7, 2008, 10:57 PM

    Dear Laura What's the percent of agarose? thanks a lot!
    Reply

    Posted by: AnonymousOctober 12, 2008, 9:53 PM

    I understand that the agarose has to be kept at 50C to keep it liquid - but dŒs that high temperature damage the brain cells?
    Reply

    Posted by: AnonymousJune 5, 2008, 2:44 PM

    The high temperature dŒs not seem to damage the brain cells - just make sure to put them on ice immediately after embedding.
    Reply

    Posted by: AnonymousJune 6, 2008, 4:33 PM

    Dear Laura, It is possible to culture the slices for ²-4 weeks? Thanks a lot
    Reply

    Posted by: AnonymousJune 13, 2008, 11:11 AM

    Hello, can you clarify the slice culture recipe for me in more detail - the recipe indicates Hanks BBS - is this in fact Hanks HBSS (ie. Hanks buffered salt solution)?. Thanks, Val
    Reply

    Posted by: AnonymousAugust 5, 2008, 10:16 AM

    fantastic!
    Reply

    Posted by: AnonymousOctober 3, 2008, 6:21 AM

    Dear Laura, First of all, tahnk you very much for the video, really explicative. Just two cuestions: - Do yo know whether the agarose could interfere in RNA extraction from the slices? - Any special consideration in order to remove the meninges? Than you very much, Alejandro García University of Salamanca (Spain)
    Reply

    Posted by: AnonymousDecember 8, 2008, 7:01 AM

    Dear Dr. Laura  i want to know if the same procedure will be applied to culture adult brain tissue.
    Reply

    Posted by: AnonymousDecember 12, 2008, 6:06 AM

    Dear Laura,   Can E18 cortical neurons be freezed and thawed? if so, for how long can one freeze them and still work with them? Can you recommend me a specific freezing/thawing protocol?   Thanks, Alex A.
    Reply

    Posted by: AnonymousMarch 7, 2009, 11:00 AM

    Dear Laura,

    Thanks for the nice video. I was curious what steps you take to minimize the risk of bacterial contamination of your slices.

    Nick Mellen
    Pediatrics
    University of Louisville
    Reply

    Posted by: Nicholas M.June 3, 2009, 3:29 PM

    Dear Laura,

    That was an exceptionally helpful tutorial. I was wondering what settings you used for the vibratome and what blade angle do you find to be most effective? I've been trying to adapt the protocol for E13.5 mice and have had difficulty making clean cuts without tearing the slice.

    Thanks so much.

    Cary Fu
    Vanderbilt University
    Reply

    Posted by: Cary F.August 6, 2009, 9:20 PM

    It's very interesting content.
    Reply

    Posted by: AnonymousNovember 23, 2009, 9:27 PM

    Hello Laura,
    Thanks for this descriptive technique video. I have a couple of questions:
    1. Some people use a Tissue Chopper/Slicer to make 300-400um slices and do not use the agarose embedding technique (mostly for postnatal slices). I would like to start making P7 cerebellal organotypic cultures and am evaluating my options. I have ready access to tissue chopper and was wondering if you have thoughts on this. As lastly, do you do ICC after fixing the slices?

    Thanks much in advance!
    Monika
    Reply

    Posted by: Monika D.January 29, 2010, 1:51 PM

    Dear Laura,

    I also wonder that how can you ensure germ free ?
    Reply

    Posted by: AnonymousOctober 22, 2011, 2:06 AM

    Dear Laura,
    I have a question with respect to organotypic slice culture. We have performed the migration study of cancer stem cell on brain xenograft model using organotypic slice culture. Also, to observe the migration of cancer stem cell, we have used to Live imaging machine. After set-up of slice culture onto the machine (37 and 5% CO²), our whole brain or our fluorescence-labelled cancer stem cell were saturated as green at 4hours after set-up. Because of this phenomenon, we can not observe cell movement. Why? Is this autofluorescence by damage of brain on preparation?
    Reply

    Posted by: AnonymousDecember 17, 2012, 9:57 PM

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