[Histonet] bone/cartilage sections falling off slides ISH

From:"Nicole Collette"


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

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