[Histonet] Re: HELP! "alien" bubbles ?

From:Gayle Callis


Two possibilities here:

1)  Is the Cytoseal 60 in a plastic bottle that has the tiny lift up tab at 
top so you can dispense?  If so, anytime time I have used these bottles for 
coverslipping, I get bubbles.  When this bottle is tipped back and forth, 
you not only agitate the media, but the top allows air to get sucked into 
the media i.e. regurgitation?  You can tip the bottle on its side to ensure 
the tip is always filled with media and no air gets back in, although this 
leads to leakage of media from tip - too messy!

  OR  Use the old fashioned method (our favorite - a drop falling off of 
either a narrow glass rod, or applicator stick onto the section.).  If you 
use applicator stick, it can be tossed when done.   Just pour media into a 
bottle with tight lid.  OR use the classic balsam bottle with glass rod - 
you can still get these from Fisher.  We keep these around for huge 
staining projects.

The other possibility is incompatibility of solvents although this may be 
an old wives tale - was told milleniums ago that using toluene based 
mounting media with sections coming out of xylene will cause bubbles - 
although we do not have many problems doing just this in our lab.

If coverslipping out of a xylene substitute, i.e. Clearite 3, we make sure 
we have a mounting media compatible with that solvent.  Check with 
manufacturer.   Most mounting medias seem to be toluene based.  If we need 
to thin a thickened media, we use the solvent it is dissolved in. ie. 
toluene, NOT xylene.  Our Richard Allan toluene based mounting media is 
compatible with xylene and Clearite 3. Propar xylene substitutes, but we do 
use "drop off a stick"  method to avoid bubbles.

Instead of adding more dehydrants, add extra clearing station and be sure 
to coverslip out of one that is not close to dehydrant.   We use 2 X 
clearing, and coverslip out of the 3rd clearant.  If the sections are 
thicker, slightly longer times in dehyrants and clearing agents may be 

  My overall personal opinion is that goofy dispenser bottle is the 
culprit, and they are NOT used here anymore because we dislike them 
intensely.  This is also a problem with aqueous mounting media dispenser 
bottles - we lay these on their side to keep tip filled and not with air.

At 09:49 AM 4/5/2005, you wrote:
>I have just completed an immuno rxn on rabbit brain and have come up
>with micro-bubbles that cant be de-hydrated?  I mounted and stained one
>set of tissue with cresyl violet.  No rxn was done on this set and they
>are perfect.  Next day, I ran 2 sets through immunos.  One with BDA and
>the other a BDA/PHAL .   Dehydrated as usual , mounted with Cytoseal 60
>and checking it under the scope (because these bubbles can not be seen
>while coverslipping) the sections are "surrounded"  by bubbles and on
>the perpherals of the tissue but none on the inner cells?  Repeated a
>couple of slides with a longer dehydration step and it made NO
>difference.  I would expect to see 'less' of the bubbles but it was
>unchanged?   Anyone have any idea what is going on??
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