Re: [Histonet] need advice badly

From:Bryan Llewellyn

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.

Bryan Llewellyn

----- Original Message ----- 
From: "N Fournier" 
Sent: Tuesday, November 21, 2006 3:16 PM
Subject: [Histonet] need advice badly

Dear Colleagues,

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,

N Fournier

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