Could you try something for me? Could you try post fixing the 45 degree
section is something? Take two 45 degree sections treated exactly the same
and post fix one in maybe mercury. Then stain both of them; if the formalin
one has polka dots and the mercury one doesn't you have some answer. If both
have polka dots, then I'm stumped. I'm just assuming it is the effect of
extra heat on poorly fixed tissue that is stopping the mordanting effect of
aluminium ions on the nucleic acids; a touch of mercury may be the solution.

If it works, then fix in formalin longer; sound sense?

-----Original Message-----
From: Rebecca Barnhart [mailto:RBARNHART@summithealth.org] 
Sent: 14 February 2005 16:06
To: histonet@lists.utsouthwestern.edu
Subject: [Possible SPAM] - RE: [Histonet] Polka dots and RE: Water
bathtemperature[Scanned] - Found word(s)[Scanned]

Yes the waterbath is cleaned everyday and there is no wax left on it.  I am
sure that all the wax is being removed or we would see it on both section
(from the waterbath at 38 and 45).  I have done test time after time
staining several slides (some cut at 38 and others at 45) at the same time
and the slides cut at 45 will always show polka dots, even with fresh
americlear (we do not use xylene) and alcohols.  

>>> Kemlo Rogerson  2/14/2005 11:01:29 AM >>>
But why does it happen only with the 'hotter' bath? Surely the wax would not
be removed from the sections floated out on the 'cooler' bath as well? You
would assume that it is either the effect of the 'extra' heat or that there
is something odd about that waterbath. I assume that the bath is clean and
we are not dealing with wax melting in the 'hotter' bath and thereby
contaminating the sections?

-----Original Message-----
From: Gayle Callis [mailto:gcallis@montana.edu] 
Sent: 14 February 2005 15:43
To: JOHN PHILLIPS; Histonet@lists.utsouthwestern.edu 
Subject: [Histonet] Polka dots and RE: Water bath temperature[Scanned]

I agree with John on this polka dot problem.  Also, stirring your paraffin 
redistributes polymer and other additives.  This should be done just before 
embedding EACH day insure these polymers have not settled to the bottom 
into little blobs or solid polka dots that may not be totally removed by 
xylenes.  In addition to increasing time in xylene, you can also add one 
more container of xylene or xylene substitute and change the first absolute 
alcohol after xylene more often to prevent excessive carry over of paraffin 
residues.

A quick test for rehydration solvents. Take a pasteur pipette and sample 
each alcohol change after xylene.  Squirt this sample into water.  If it 
turns milky white, then you have carry over of xylene and paraffin into the 
alcohols, and need to rotate out all changes more frequently.  If this 
happens in the 95% just before 70% and water, you really have trouble with 
paraffin residues removal.



At 02:42 AM 2/14/2005, you wrote:
>The problem with staining may be due to unsatisfactory removal of the wax 
>prior to staining for H&E. I think you are looking to the wrong place for 
>the solution to your problem. Try extending your dewax time in xylene 
>before you hydrate your sections on the staining run. If the problem goes 
>away PERMANANTLY then you have your answer. Good luck.
>
>John,
>Wrexham,Wales.U.K.
>
>-----Original Message-----
>From: Rebecca Barnhart [mailto:RBARNHART@summithealth.org] 
>Sent: 11 February 2005 19:24
>To: histonet@lists.utsouthwestern.edu 
>Subject: [Histonet] Water bath temperature
>
>
>We were having a problem that the tissue would not take up the
>hematoxlyin but would take up the eosin but only in some areas, so it
>looked like polka dots.  We started one by one changing solution trying
>to figure out where it was coming from.  We had all fresh solutions and
>still had polka dots.  I tried the only other thing left to change, I
>lowered the temperature of the water bath from 45 to 38 and this worked.
>  We currently are using ParaPlast Xtra with a melting point of 52.  We
>have all tried figure out why the temperature in the water bath will
>make the difference.  Several times I have cut the same tissue at
>different temperatures ranging from 38 - 45 and every time there are
>polka dots at the higher temperatures.  Does anyone have any idea why
>this would be?
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)



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