Gallyas F, Wolff J R, Böttcher H, Zaborszky L
Stain Technol. 1980 Sep;55(5):299-306. doi: 10.3109/10520298009067258.
After reconsidering the physicochemical mechanisms involved in the so-called degeneration methods for the demonstration of axons and nerve terminals, the method of Eager was fundamentally modified in order to stabilize the staining process. This resulted in a simple and reliable method which stains degenerating terminals and lysosomes with a high degree of selectivity and sensitivity. Frozen sections 30 to 50 micrometers thick are prepared from material fixed with formaldehyde by cardiac perfusion. The staining procedure consists of 5 steps: 1) alkaline pretreatment (pH 13), 2) silver impregnation, 3) washing, 4) development at pH 5.0-5.5 monitored by an indicator, and 5) washing in acetic acid. Possible faults can be easily detected by their specific effects on the staining results. Primary submicroscopic silver precipitates are localized selectively in the osmiophilic parts of lysosomes and those degenerating presynaptic elements that are surrounded by glial processes. In degenerating axons, precipitates originating from mitochondria can usually be distinguished from terminal degeneration by their different size, shape, or characteristic arrangement. Nonspecific staining is restricted to glial fibrils, erythrocytes, and single cell nuclei. Dark field illumination can be applied routinely and television image analysis can be used for quantitative evaluation because of low background staining.
在重新考虑了用于显示轴突和神经终末的所谓变性方法所涉及的物理化学机制后，对伊格尔（Eager）方法进行了根本性的改进，以稳定染色过程。这产生了一种简单可靠的方法，该方法能以高度的选择性和敏感性对变性终末和溶酶体进行染色。用甲醛通过心脏灌注固定的材料制备30至50微米厚的冰冻切片。染色程序包括5个步骤：1）碱性预处理（pH 13），2）银浸染，3）洗涤，4）在pH 5.0 - 5.5下用指示剂监测显影，5）在乙酸中洗涤。通过它们对染色结果的特定影响，可以很容易地检测出可能的错误。初级亚显微银沉淀选择性地定位于溶酶体的嗜锇部分以及那些被神经胶质细胞突起包围的变性突触前成分中。在变性轴突中，源自线粒体的沉淀通常可以通过其不同的大小、形状或特征性排列与终末变性区分开来。非特异性染色仅限于神经胶质纤维、红细胞和单个细胞核。由于背景染色低，暗视野照明可常规应用，电视图像分析可用于定量评估。
