[Histonet] bone/cartilage sections falling off slides ISH

[Liz Chlipala]

Collette

For bone and cartilage I would try to work with enzymatic retreival methods,
such as proteinase K, pronase, trypsin, or hyaluronic acid.  Not all
antibodies will work with enzyme digestion but quite a few do, the enzyme
retreival methods will not be as harsh as HEIR.  If you do have to use HIER
I would use a steamer or a gentler method.  Lower temperatures with longer
retreival periods might help, but I have been unable to get these to work
well in my hands on bone.  Pasty I think has had success with longer
retrieval times at lower temps, maybe she can respond with some suggestions.
I also use a good silane treated slide, and there are ones that are better
than others.  When I section I do not let bone sections air dry at room temp
I place them on a warm hot plate (around 44 degrees) you don't want the
paraffin to melt, I find that that sometimes causes the articular cartilage
to flip.  I leave then on the hot plate overnight and do not place them in
an over afterwords.  I get pretty good success with joint sections from
mice, rats, dog, sheep, goat, etc. with this method.

Liz 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
P.O. Box 18592
Boulder, CO 80308
phone (303) 735-5001
fax (303) 735-3540
liz <@t> premierlab.com
www.premierlab.com
 
Ship to Address:
 
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University of Colorado at Boulder
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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Nicole
Collette
Sent: Friday, March 02, 2007 12:52 PM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] bone/cartilage sections falling off slides ISH

Hello,

I am relatively new to the land of histology (you can also read that as
"self-taught"), and I am in a lab that is studying skeletal development. We
are doing some histology stains and some fluorescent ISH on embryonic mouse
limb sections, and are having trouble keeping the sections on the slides
during processing.

The tissues are embryonic mouse tissues, E14.6, E16.5, fixed in 4% PFA,
embedded in paraffin using standard protocols. I have used charged slides
and polylysine slides and in both cases, I float the ribbons in DEPC
water/Ethanol and then move them to 39C water bath for putting on slides
(brand new, accurate temperature, no water bath additives, clean DEPC
water). I do not seem to have issues for the most part with the cartilage
wrinkling up, which can sometimes be an issue. I dry the slides in vertical
position O/N and then store @ -20C with dessicant.

The sections stick just fine during regular staining (H&E, cartilage
stains), and anything without bone sticks just fine during any processing
(taken from the same embryos at the same time, embedded the same way), but
the sections that contain only limbs fall right off. I am wondering if this
is a mechanical issue related to doing steps in coplin jars with shaking as
opposed to on individual horizontally situated slides, a processing issue
pre-ISH, or some other handling issue that might be a simple case of
"newbie-don't know".

The tech I have doing the ISH does a relatively lengthy hot
antigen-retrieval step at the beginning that I think may be the problem
(they swear by it, but I think it's a bad idea for ISH), but she says even
without that step, the sections don't fare well. The RNA quality is good,
when we get sections that stick, the probes work well. The sections come off
primarily starting from the middle of the long bone cavity working out
toward the cartilage edges.  I think there may also be an issue of the Prot
K step, since their protocol is optimized for whole body sections at the
same stage, so there is a much larger piece of tissue on the slide (we are
working on doing control experiments for these options now). I just want to
see if there is something else I can be doing to keep the slides together, I
know that bone and cartilage are "exception" tissues, and different rules
may apply when handling/processing. Any advice would be most gratefully
appreciated.

Thanks in advance for the resounding response! (and sorry about the lengthy
description, wanted to provide as much info as possible)
Sincerely,
Nicole Collette
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