[Histonet] need advice badly

[N Fournier]

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 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 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