[Histonet] Xylene substitutes in staining line

Gayle Callis gayle.callis at bresnan.net
Thu Jun 25 14:33:16 CDT 2015

You wrote: 


Hello Histo Land,

Does anyone use xylene substitutes in their stain line only? I am especially
interested in d-limonene options. Thanks in advance!




We used xylene substitutes in the stain line for both deparaffinization and
dehydration but did not use limonene (citrus smelling).   We preferred the
Clearite 3 (Richard Allan) i.e. single aliphatic hydrocarbons  also
available from other vendors, or Propar (Anatech)  for this purpose.
Limonene was particularly offensive and personally made me turn green with
nausea!  It was banned from my lab due to intolerance to the smell which
also included banning limonene based household cleaners in my home.    I
think people can become sensitized to this substitute so be careful about
handling and working under a hood.   We also used Clearite 3 for tissue
processing to eliminate xylene.   


There are caveats about using xylene substitutes.  They are sensitive to
residual water carry over and do not clear water as well as xylene.  We
added an extra stations to deparaffinize sections and at the end of
dehydration sequence ensure NO residual water carry over before mounting a
cover glass.  On some high humidity days, Clearite 3 would be cloudy meaning
the last stations with Clearite had to be changed to fresh solvent not
exposed to humid air.  To combat a water carryover problem commonly not seen
with xylene and its ability to handle water carryover better, we rotated the
substitutes frequently during a work week so the last station was fresh.
If you have cloudy looking sections after cover slipping from a xylene
substitute, then you have water carry over.  A hint is if the last alcohol
in the dehydration series is pink, then there is water carryover.   Due
diligence is necessary to avoid poor paraffin removal and also good
dehydration.   In a very busy laboratory, this means extra time and expense
to deal with a xylene substitute.    


The test for paraffin carry over in  deparaffinization sequence, one can
pipette a few mls of last 95% alcohol (closest to water) into a glass beaker
of tap water.  It the aliquot turns the water cloudy, then you have paraffin
carry over into the alcohol.  If not seen in last 95%, the test the 95%
station by working backwards in the deparaffinizing sequence.  If any
alcohol test is cloudy, then that alcohol and all previous stations were
changed in our lab.  Some rotation of clear test was done into an earlier
station slot. This will test also works when using xylene.  

However, I really like the emerging practice of using hot soapy water for
deparaffinizing sections recently discussed in depth on Histonet.   There is
a wonderful publication by Tony Henwood et al,  J of Histotechnol 2013;
36(2):45-50.  Tresa Goins also entered in on this discussion about their
lab's success with this method.  Go to Histonet Archives and read the
commentary.   It certainly means less dependence on organic solvents, less
exposure to potentially toxic chemicals, and the expense of solvent
disposal.   I think the soap method is definitely worth trying. 

Hope this helps

Gayle M. Callis  HTL/HT/MT(ASCP)  



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