Re: long term paraffin section storage/tissue array construction/slide labeling instrumentation

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From:Mary Ann Miller <> (by way of histonet)
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Dear Liz, and others

Our lab also works with hundreds and hundreds of different tumors since we
are the repository for several SWOG GI protocols. We have attempted to deal
with these issues for many years. We set up an experiment in 1996 whereby
we studied several different tumors known to have differing intensities of
staining on freshly cut slides. Tissues were cut on Evergreen ++ slides and
then studied at monthly intervals for 2 years. We routinely cut our tissues
using a 42C waterbath containing no additives. We then allow the slides to
dry at RT for at least 24 hours before stacking and filing for later use.
For this study we coated half the slides with paraffin and left the other
half plain.The results were variable but mostly block dependent.(We had no
knowledge of fixation techniques since the blocks came from 50 different
places.)  The slides coated with paraffin actually lost far more activity
over time than those simply stored at RT in old Shandon filter card boxes.
They also took up much more storage space. We abandoned this method. The
antibodies we specifically studied for this experiment were P-53, WAF-1 and
Ki67. In addition we have checked many other antibodies over time. We have
found our results to be dependent on how carefully the slides were
originally cut and on the individual antibody.(could also be the antigen of
course). A good number of the antibodies we use seem to remain stable over
at least several years. For the most part staining intensity may go from 4+
to 3+ but is still quite acceptable. The problem is is of course worse if
the initial stain is only 2+. One other thing I should also note,  we use
the Ventana ES Immunostainer.

I'm not quite sure what you mean by tumor arrays but we routinely make up
what we call our tumor sausage blocks. For these we take small (2-3mm)
pieces of "old" tumors from our surgical files, melt them down and reembed
8-10 of these pieces in one larger block. We have colon, breast, lung
sausages etc. This number is plenty to show variability across tissues. We
then cut 10-20 slides at a time and store them as above for use as positive

One more thing --if you receive replies directed only to you could you
please share them with me? I hate to miss important information.

Sincerely,    Mary Ann

At 09:08 PM 12/15/99 -0800, you wrote:
>Hello Fellow Histonet Folks,
>I am trying to gather information regarding paraffin section storage
>systems, tissue array creation methods and glass slide labeling
>instrumentation.  Any knowledge that individuals would kindly be willing to
>contribute to the questions listed below would be very much appreciated.
>1. I have been following the discussions of the past few weeks regarding
>long term paraffin section storage methods.  We are particularly interested
>in the method mentioned by Barry Rittman  which involved using a thin
>coating of paraffin to cover the tissue section.  Could Barry or any one
>else familiar with this method please provide me with any additional info
>regarding the technique?  How long can one store tissue sections using this
>method without compromising antigenicity?  Does this technique prevent
>oxygen from reaching the tissue?
>We are currently using a nitrogen storage system for storage but, for the
>large number of slides/cases we store, it is a very inefficient system
>because we are constantly going in and out of the storage boxes thereby
>intermittently exposing the sections to oxygen.  Our system consists of
>storing the paraffin sections in black slide boxes which are placed in
>double zip lock bags that are then filled with nitrogen and stored at 2-8C.
>Does anyone have a more efficient nitrogen storage system in place?
>2.  We have access to a vast number of tumor paraffin blocks that are
>available to us for only a short time period.  It would be ideal to have the
>capacity to construct a tissue array or, in other words, a paraffin block
>that contains a large number (1000) of tumors.  In this way the original
>specimens would not be compromised and we would have access to a large
>source of tumors that could be characterized immunocytochemically in an
>efficient manner.  These arrays seem to be different then Battifora tissue
>rolls, which have been previously described, in that the specimens used to
>make the array are smaller in size, larger in number and arranged in a very
>organized grid like pattern.  Although, I do beleive that some of the same
>pitfalls exist in cutting and evaluating both the array and tissue rolI.  I
>have read that the National Institute of Health has a technique for
>constructing these tissue arrays using specialized instruments and have also
>heard that other groups are creating them.  Does anyone have experience with
>this process or any further information?
>3.  Is anyone using an automatic slide labeler that they are happy with?  We
>would like to have the ability to, for example, input case/lab number with
>block id and maybe a date which could then be imprinted on a predetermined
>number of glass slides.  A labeler that imprints "paper" labels would also
>be considered . Vendors responses to this subject are welcome.
>I apologize in advance if there have already been discussions of these
>topics on the histonet.  I attempted to perform the initial research on the
>histonet archives but was unsuccessful because the archives are down and
>will not be available again until next year.  With the immense pool of
>knowledge on the histonet, I am hopeful that at least a few of my questions
>will be answered -- thanks!
>Elizabeth Donato
>Cancer Biology
>Fred Hutchinson Cancer Research Center
>Ph:   206-667-4501
>Fax: 206-667-5815
Mary Ann Miller
University of Cincinnati
College of Medicine  ML 529
231 Bethesda Avenue
Cincinnati, Ohio  45267
FAX: (513) 558-2289

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