[Histonet] Re: Holes in paraffin, remedies

[gayle callis]

Hugh wrote: 

 

Sophia,

 

Holes like you mentioned, are probably from the polymers or impurities in
your embedding wax separating.  Gayle Callus mentioned this several times
(see histosearch), and recommended "Stirring" the embedding paraffin every
so often (before every use).  It could also be your tissue processing.
Check to make sure it is properly processed.  Embed without letting the wax
solidify before doing so.  You may have a bad batch of reagents?

 

As for the stainer, Hazy nuclei, as long as you are sure your hemotoxylin is
fine, are indicative of incomplete deparaffinzation, which could mean your
OVEN or your reagents.  It could also be your acid/base solutions.

 

Note:  Have you started a new lot # of wax?  How about xylene
(xylene-substiture) or alcohols?  I have heard people saying X-brand
paraffin was having problems with impurities that could cause these
problems.  I also heard it was solved last year. 

 

Also, your stainer has very little leeway in deparaffinzation.  You might
have to do things by hand until this problem is resolved.  You know,
deparaffinize to water using the longer method, then finish the H&E in the
stainer.  Does this help?

 

 

Good luck (whoa, you have your work cut out for you),

 

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Hugh, 

 

Thank you for the kind words and your reply was right on the money.    In
fact, we stirred the paraffin daily before embedding, since the settling of
polymers is not just occasional.    Also, clean your embedding center
frequently, before adding more paraffin.   

 

There is a clever little test for paraffin carryover into your rehydration
alcohols when removing paraffin.   1)  Use a glass beaker, add a few ml of
used alcohol  starting with last 95% just before 70% in deparaffinization
setup.   2) Pipette a few mls of  tap water into this aliquot of 95% and
look for cloudiness.   If a white cloud occurs, you have paraffin carryover
all the way down your deparaffinization process.   3) If you don't see
cloudy in this last 95%, test the 95% before this one, and keep going
backwards towards the xylene or xylene substitute.  If the next 95% is
cloudy, there is paraffin carryover.  Change out that alcohol station and
all the ones before that including the xylene/xylene substitute. 

 

Also, you can do "rotation" where you move the second ( closest to water)
95% into first 95% spot, then replace the second 95% with fresh, replace all
100% and xylene/xyl sub  as these latter are probably heavily contaminated
with paraffin which is carrying over into your alcohols at some level.   We
used two or three xylene/xylene substitute changes (three preferred when
doing IHC at 5 min/change) , two (or three)  100%, two 95% and one 70%
alcohol changes before distilled water, 3 minutes per change, with hand
staining.    Change distilled water frequently, fresh daily and more changes
when staining many slides as you don't need alcohol carry over into your
hematoxylin.   Careful monitoring of your solvents and distilled water
should allow better hematoxylin staining. 

 

We also changed our acidic solution after hematoxylin and bluing solutions
daily, these solutions are cheap plus always doing a 1 minute running water
rinse after hematoxylin, acidic solution, and bluing.    If you don't have
running water rinsing, at least change your water rinses before each
staining run.  

 

This all sounds very picky, but our H&E staining was very successful.  

 

Gayle Callis

HTL/HT/MT(ASCP)