sectioning


Sun Apr 3 02:14:00 EST 2005


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Hi everyone,

We section in situ'd embryos after embedding in JB4 resin and get nice
results with really good morphology.  We cut our embryos on a motorized
microtome in 4-5 micron sections.  After we take pictures of our in situs,
we follow over the same sections with an H&E stain to see the histology if
we want.  With a strong stain, you can still see the in situ over the H&E.

I'd be happy to give our protocol to anyone who would like to see it.

Mary, we'd be interested in your paraffin protocols.  Have you tried
antibody staining after sectioning?

Thanks,
Becky


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Rebecca Burdine, Ph.D.
Assistant Professor
Department of Molecular Biology
Washington Road
Princeton University
Princeton, NJ 08544

Ph  (609) 258-7515
Fax (609) 258-6730
e-mail: rburdine at molbio.princeton.edu

-----Original Message-----
From: owner-zbrafish at hgmp.mrc.ac.uk [mailto:owner-zbrafish at hgmp.mrc.ac.uk]
On Behalf Of ""
Sent: Friday, April 01, 2005 9:53 AM
To: zbrafish at net.bio.net
Subject: RE: sectioning

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Jeffrey,
I section both embryos and adult fish embedded in paraffin. I would be
happy to share my embedding protocol with you, please let me know. I have
not had vast experience yet with whole mount in situs, my experience
yielded me chewed up over digested skeletons, and not much else. I will be
revisiting this procedure in the near future, and any pearls of advice
would be greatly appreciated.
Also we are in the pilot phase of a study in which we will need to isolate
adult zebra fish tissues for RNA isolation, is there anyone who is doing
this that I might be able to chat with?
Cheers!

Mary Georger
Cardiovascular Research Institute
University of Rochester Medical Center
KMRBX 2-11301
575 Elmwood Avenue
Rochester, NY 14642
585-273-1548

War may sometimes be a necessary evil.  But no matter how necessary, it is
always an evil, never a good.  We will not learn how to live together in
peace by killing each other's children.  President Jimmy Carter


> ----------
> From: 	"Jeffrey Hannah"
> Sent: 	Friday, April 1, 2005 7:39 AM
> To: 	zbrafish at net.bio.net
> Subject: 	sectioning
>
> ---------------------------------------------------------------------- ----
>
> Hello everyone,
>   After whole-mount in situs, I often section my zebrafish after
> embedding them in paraffin.  Despite the fact that I follow the same
protocol and use the same sectioning methods for all the embryos, many
times there are problems with some of them.  My most common problem is
that the embryos will often crumble and crack.  I have tried isolating the
problem, but I can't seem to figure it out.  Does anyone use
> paraffin for zebrafish sections?  If so, have you run into this  problem
and could I see your protocol?  Do plastic or cryogenic sectioning work
better for whole-mount in situ zebrafish?  Or am I better off performing
in situ hybridization after sectioning?  Any advice is appreciated.
>
> Jeffrey Hannah
> Children's Hospital of Philadelphia
>
> ---
>
>

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