Abstract
Artifacts from autolysis can be a problem in retrospective flow-cytometric analyses of DNA content in paraffin-embedded tissues. Autolyzed tissue from rat liver, human liver, and rat spleen were stained for DNA and nuclear protein to determine if this technique would be useful in identifying partially degraded cells. After the tissue was deparaffinized and rehydrated, the nuclei were isolated using 0.5% pepsin. Propidium iodide (PI) and fluorescein isothiocyanate (FITC) were used to stain DNA and nuclear protein. When unfixed rat liver tissue was allowed to undergo autolysis at 4°C for 24-48 b before fixation, there was a progressive broadening of the G1 and G2M DNA peaks and a slight increase in the average DNA contents of these peaks. Nuclei that stained more intensely with PI also stained more intensely with FITC. Similar results were obtained using human liver and rat spleen. Sometimes the increased PI staining resulted in a false aneuploid peak. The distinctive skewing of the DNA/nuclear protein histograms from autolysis was reduced by increasing the incubation of the tissue in 0.5% pepsin from 0.5 h to 1.5 h during the nuclei-isolation step. The DNA/nuclear protein method provides a means for identifying artifacts from autolysis, whereas the extended pepsin treatment provides a means for reducing these artifacts.
| Original language | English |
|---|---|
| Pages (from-to) | 565-568 |
| Number of pages | 4 |
| Journal | Cytometry |
| Volume | 14 |
| Issue number | 5 |
| State | Published - Jan 1 1993 |
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Keywords
- autolysis
- flow cytometry
- nuclear protein
- paraffin-embedded tissue
ASJC Scopus subject areas
- Biophysics
- Cell Biology
- Endocrinology
- Hematology
- Pathology and Forensic Medicine
Cite this
Recognition and reduction of artifacts from autolysis in paraffin-embedded tissue using DNA/nuclear protein flow cytometry. / Pollack, Alan; Ciancio, Gaetano; Terry, N. H A; Block, Norman L.
In: Cytometry, Vol. 14, No. 5, 01.01.1993, p. 565-568.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Recognition and reduction of artifacts from autolysis in paraffin-embedded tissue using DNA/nuclear protein flow cytometry
AU - Pollack, Alan
AU - Ciancio, Gaetano
AU - Terry, N. H A
AU - Block, Norman L
PY - 1993/1/1
Y1 - 1993/1/1
N2 - Artifacts from autolysis can be a problem in retrospective flow-cytometric analyses of DNA content in paraffin-embedded tissues. Autolyzed tissue from rat liver, human liver, and rat spleen were stained for DNA and nuclear protein to determine if this technique would be useful in identifying partially degraded cells. After the tissue was deparaffinized and rehydrated, the nuclei were isolated using 0.5% pepsin. Propidium iodide (PI) and fluorescein isothiocyanate (FITC) were used to stain DNA and nuclear protein. When unfixed rat liver tissue was allowed to undergo autolysis at 4°C for 24-48 b before fixation, there was a progressive broadening of the G1 and G2M DNA peaks and a slight increase in the average DNA contents of these peaks. Nuclei that stained more intensely with PI also stained more intensely with FITC. Similar results were obtained using human liver and rat spleen. Sometimes the increased PI staining resulted in a false aneuploid peak. The distinctive skewing of the DNA/nuclear protein histograms from autolysis was reduced by increasing the incubation of the tissue in 0.5% pepsin from 0.5 h to 1.5 h during the nuclei-isolation step. The DNA/nuclear protein method provides a means for identifying artifacts from autolysis, whereas the extended pepsin treatment provides a means for reducing these artifacts.
AB - Artifacts from autolysis can be a problem in retrospective flow-cytometric analyses of DNA content in paraffin-embedded tissues. Autolyzed tissue from rat liver, human liver, and rat spleen were stained for DNA and nuclear protein to determine if this technique would be useful in identifying partially degraded cells. After the tissue was deparaffinized and rehydrated, the nuclei were isolated using 0.5% pepsin. Propidium iodide (PI) and fluorescein isothiocyanate (FITC) were used to stain DNA and nuclear protein. When unfixed rat liver tissue was allowed to undergo autolysis at 4°C for 24-48 b before fixation, there was a progressive broadening of the G1 and G2M DNA peaks and a slight increase in the average DNA contents of these peaks. Nuclei that stained more intensely with PI also stained more intensely with FITC. Similar results were obtained using human liver and rat spleen. Sometimes the increased PI staining resulted in a false aneuploid peak. The distinctive skewing of the DNA/nuclear protein histograms from autolysis was reduced by increasing the incubation of the tissue in 0.5% pepsin from 0.5 h to 1.5 h during the nuclei-isolation step. The DNA/nuclear protein method provides a means for identifying artifacts from autolysis, whereas the extended pepsin treatment provides a means for reducing these artifacts.
KW - autolysis
KW - flow cytometry
KW - nuclear protein
KW - paraffin-embedded tissue
UR - http://www.scopus.com/inward/record.url?scp=0027319525&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027319525&partnerID=8YFLogxK
M3 - Article
C2 - 8354130
AN - SCOPUS:0027319525
VL - 14
SP - 565
EP - 568
JO - Cytometry Part B - Clinical Cytometry
JF - Cytometry Part B - Clinical Cytometry
SN - 1552-4949
IS - 5
ER -