Van Prooijen-Knegt A C, Redi C A, Van der Ploeg M
Histochemistry. 1980;69(1):1-17. doi: 10.1007/BF00508362.
Quantitative aspects of DNA losses during fixation and pararosaniline(SO2)-Feulgen staining of microscopic preparations were studied. The preparation of a new cytochemical model, consisting of DNA-protein layers (with thicknesses between 0.1 and 5.0 micrometer) on microscopic glass slides is described and potentialities and limitations of this model are discussed. Polyacrylamide films into which high molecular weight calf thymus DNA or chicken erythrocyte nuclei had been constrained served as another model. As biological objects chicken erythrocyte nuclei and rat liver nuclei either in suspension or on microscopical glass slides were used. The experimental results indicate a loss of about 5% of the DNA due to the fixation procedure applied. Hydrolysis in 5 N HCl at room temperature, staining with the pararosaniline-Schiff medium and rinsing with sulfurous acid induced losses of DNA too, varying in amount depending on the type of preparation used. About 10% of the original DNA content is lost in total from chicken erythrocyte nuclei and rat liver nuclei dried on microscopical glass slides, from chicken erythrocyte nuclei constrained in polyacrylamide films, and from DNA-protein layers on microscopic glass slides. For nuclei fixed and stained in suspension the total losses amount to about 40%. The differences in losses between various types of preparations are discussed. Biochemically, the content of DNA originally present per chicken erythrocyte nucleus was determined to be 2.52 pg, a value, which is in good accordance with reliable biochemical data published already. It is shown that calibration of cytochemical staining intensities into biochemical units or absolute amounts of material by use of a model system, is only reliable when it is known or to be expected that both the loss of material due to fixation and staining, and the stoichiometric relation between material present and dye molecules is identical. The same holds for the application of internal biological reference systems.
研究了显微镜标本固定及对品红（SO₂）-福尔根染色过程中DNA损失的定量方面。描述了一种新的细胞化学模型的制备方法，该模型由显微镜载玻片上的DNA-蛋白质层（厚度在0.1至5.0微米之间）组成，并讨论了该模型的潜力和局限性。另一个模型是将高分子量小牛胸腺DNA或鸡红细胞核限制在其中的聚丙烯酰胺膜。使用悬浮状态或显微镜载玻片上的鸡红细胞核和大鼠肝核作为生物对象。实验结果表明，由于所采用的固定程序，DNA损失约5%。在室温下于5N HCl中水解、用品红-席夫培养基染色以及用亚硫酸冲洗也会导致DNA损失，损失量因所用标本类型而异。在显微镜载玻片上干燥的鸡红细胞核和大鼠肝核、聚丙烯酰胺膜中限制的鸡红细胞核以及显微镜载玻片上的DNA-蛋白质层中，原始DNA含量总共损失约10%。对于在悬浮状态下固定和染色的细胞核，总损失约为40%。讨论了不同类型标本损失的差异。从生物化学角度测定，每个鸡红细胞核中最初存在的DNA含量为2.52 pg，该值与已发表的可靠生物化学数据高度一致。结果表明，只有当已知或预期固定和染色导致的物质损失以及存在的物质与染料分子之间的化学计量关系相同时，使用模型系统将细胞化学染色强度校准为生物化学单位或物质的绝对量才是可靠的。内部生物参考系统的应用也是如此。
