Re: [Histonet] need advice badly
A common practice with hand mounted slides is to dip them in xylene, wipe
the back, wick an edge, but leave a considerable amount of xylene over the
tissue and slide. This is to facilitate the flow of mounting medium and
reduce bubbles being formed as the medium flows over the slide. However, if
there is too much xylene it dilutes the mounting medium, and while it makes
for a nicely coverslipped section, as the xylene evaporates over time the
medium contracts back, often from the tissue edge, forming a bubble.
The solution is to drain almost all of the xylene from the slide before
coverslipping. The tissue should be close to dry, but just moist. This
involves using a fair bit more mounting medium and requires some practice as
the medium flows more slowly. It is not a difficult skill to develop,
however. You could also use DPX and apply a fair amount of it so that as
the xylene evaporates it draws from the excess as a reservoir. After a
month you can strip the excess DPX off.
I prefer the first way, just be careful not to let the tissue get so devoid
of xylene that it actually does dry and starts cracking.
----- Original Message -----
From: "N Fournier"
Sent: Tuesday, November 21, 2006 3:16 PM
Subject: [Histonet] need advice badly
We are having some problems with very large air bubbles being deposited
along entire rat brain sections that were mounted onto you slides and would
like some suggestions to fix the problem. The slides appear in good order
for several weeks and then the problem emerges. On average we mount three
sections or so to a slide. The bubble tends to surround the entire section
for the most part sometimes, but the periphery of the bubbles do not seems
to merge or “flow together” for the most part. Nor does it seems that it
flows towards the periphery of the slide.
Although originally the coverslipping was performed by a junior lab member;
we have also noticed this occurring with more senior members. We thought
at first that it might be the mounting medium (used Entellan mounting medium
originally but now switch to Cytoseal 60 since this mounting medium does not
make as much of a mess); however, we have notice this occur with cytoseal
mounting medium. (I thought the bottle might have been old and have=20
subsequently replaced it. I personally used the new bottle and have had no
problems…yet). I do not believe it due to an tissue thickness since the
tissue was sectioned at approximately 40 to 50 microns on a vibrating=20
microtome. Another possibility that I thought is small microcracks causing
air to slowly enter the slide.
This is becoming extremely annoying since it takes an extremely long time to
generate the tissue for immunohistochemistry. It is also problematic for
quantification. I have thought of re-coverslipping these slides by placing
them in xylene for a few hours. The sections do not really look dried out
and the stain still remains at least from what I can discern. Is this the
best procedure to do or is this precious tissue lost for forever. (We
really do not want to re-do the immunos because of all the time and money
spent thus far). Is there a better technique to use?
Any help is appreciated,
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