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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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