Recognition and reduction of artifacts from autolysis in paraffin-embedded tissue using DNA/nuclear protein flow cytometry.
Recognition and reduction of artifacts from autolysis in paraffin-embedded tissue using DNA/nuclear protein flow cytometry.
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使用 DNA/核蛋白流式细胞术识别和减少石蜡包埋组织中自溶的伪影。
DOI：
10.1002/cyto.990140517
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Block,NL
中科院分区：
文献类型：
--
作者：
Pollack,A;Ciancio,G;Terry,NH;Block,NL
在对石蜡包埋组织中DNA含量的回溯性流式细胞术分析中，自溶产生的伪影可能是一个问题。对来自大鼠肝脏、人肝和大鼠脾的自溶组织进行DNA和核蛋白染色，以确定这项技术是否有助于识别部分降解的细胞。组织脱蜡复水后，用0.5%胃酶分离细胞核。用碘化丙啶(PI)和异硫氰酸荧光素(FITC)对DNA和核蛋白进行染色。未固定的大鼠肝组织在固定前于4℃自溶24-48h，G1期和G2M期DNA峰逐渐变宽，平均DNA含量略有增加。PI染色较强的细胞核也有FITC较强的染色。用人的肝脏和大鼠的脾也得到了类似的结果。PI染色增强有时会导致假的非整倍体峰。在核分离步骤中，将组织在0.5%胃蛋白酶中的孵育时间从0.5h增加到1.5h，可以减少自溶引起的DNA/核蛋白直方图的明显倾斜。DNA/核蛋白方法提供了一种从自溶中识别伪影的方法，而扩展胃酶治疗提供了一种减少这些伪影的方法。©1993 Wiley-Liss，Inc.
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 h before fixation, there was a progressive broadening of the G1and 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. © 1993 Wiley‐Liss, Inc.