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> This is a post from a colleague ...
>
> Cytometry Experts,
>
> We are doing flowcytometric analysis of nuclear DNA content and ploidity
> determination of paraffin embedded archival pathological lymphoma cases in
> dogs.We dewax the 30um sections by two changes of xyleneand dehydrate
> with sequential ethanol concentrations and distilled water.Finally we
> digest the tissue by pepsin and stain by standard PI staining method.
> We get nice diploid and aneuploid peaks,however the fluorescence of the
> deparaffinized samples is lower than in the fresh material.
>
> Questions:Is this the expected pattern of paraffin embedded tissue?
> If yes:what is the explaination?
> If not:how can we increase PI uptake in archival lymphoid tissues?
>
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The decrease in the PI staining in paraffin-embedded tissues is due to
the fixation of the tissue in formalin. The formalin causes
cross-linking between the DNA and histones which interferes with the PI
binding. We (Phil McCoy and I) have found that heating formalin-fixed
cells in PBS at 75C for at least one hour reverses the formalin effect
and restores the PI binding (Cytometry 16:351-356, 1994). We have also
found that this works with formalin-fixed, paraffin-embedded tissue if
the deparaffinized tissue is treated with trypsin rather than pepsin (in
press, Cytometry (Communications in Clinical Cytometry)). For further
details, I can be contacted directly (see below).
Roy
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W. Roy Overton, Ph.D.
Pediatric Hematology/Oncology Phone: 609-342-2894
Cooper Hospital/University Med. Ctr. Fax: 609-338-1468
3 Cooper Plaza, Suite 410 Email: overton@umdnj.edu
Camden, NJ 08103
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web