sectioning

Melissa Haendel mhaendel at uoneuro.uoregon.edu
Mon Apr 4 08:38:00 EST 2005


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Jeffrey,
I have always found that embryos in general do not section very nicely  in
paraffin.  I would recommend frozen sections or plastic sections for 
anything less than 72 hours.  The anatomy is preserved much better.  If 
your staining is robust, in situs following by sectioning is easier.  If 
not, you can do in situs on frozen sections but this is a bit more 
tricky.  I can send you a protocol if you decide this is necessary.  
Also, it seems embedding quality varies widely due both to the fix and 
also to whatever processor (or by hand) you might be using.

Mary,  I would highly recommend the TRIZOL reagent from Gibco, followed 
by cleanup with mini RNA columns from Qiagen.  You can actually store 
tissues in TRIZOL at -80.  It is nice to have a mini-homogenizer, which 
can be placed directly in the thawing TRIZOL.  This procedure gives very 
high quality RNA suitable for microarrays.

Melissa Haendel
ZFIN curation staff

Georger at hgmp.mrc.ac.uk wrote:

>--------------------------------------------------------------------------
>
>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
>>
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>>
>>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
>>
>>---
>>
>>
>>
>>
>
>---
>
>

-- 
Melissa Haendel, Ph.D.

ZFIN Scientific Curator

Zebrafish Information Network

5291 University of Oregon

Eugene, OR 97403-5291

Phone: (541) 346-5108


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