Gallyas F, Wolff J R, Böttcher H, Záborszky L
Stain Technol. 1980 Sep;55(5):291-7. doi: 10.3109/10520298009067257.
A method has been elaborated by which degenerating axons can be selectively impregnated with silver. Based on reconsideration of the physicochemical mechanisms of the degeneration methods it takes advantage of physical developers over the chemical ones. The staining procedure is applied to frozen sections of brains fixed with formol. It consists of 6 steps: (1) pretreatment with alkaline hydroxylamine, (2) washing in acetic acid, (3) impregnation in silver nitrate in the presence of ferric ions, (4) washing in citric acid, (5) physical development, and (6) washing in acetic acid. By electron microscopy silver precipitates by this method are almost entirely restricted to the cytoplasm of dense, degenerating axons, sparing mitochondria and myelin sheaths. No special expertise is required to achieve reproducible results. Large numbers of sections treated simultaneously, and large sections, can be stained uniformly. Light microscopic criteria are described which help diagnose the source of possible failures. Low background staining allows dark field illumination and television image analysis to be applied. The method works at survival times of only 3 to 5 days after axotomy. Hence, degenerating axons and axon terminals can be stained in alternating sections from the same brain using this method and another being described separately, which, using different conditions, demonstrates degenerating axon terminals.
已研发出一种方法，通过该方法可选择性地用银浸染退化的轴突。基于对退化方法物理化学机制的重新思考，该方法相较于化学显影剂更青睐物理显影剂。染色程序应用于用甲醛固定的脑冰冻切片。它包括6个步骤：（1）用碱性羟胺预处理，（2）在乙酸中洗涤，（3）在铁离子存在下用硝酸银浸染，（4）在柠檬酸中洗涤，（5）物理显影，以及（6）在乙酸中洗涤。通过电子显微镜观察，用这种方法形成的银沉淀几乎完全局限于致密的退化轴突的细胞质中，线粒体和髓鞘不受影响。获得可重复的结果不需要特殊的专业技能。同时处理大量切片以及大切片时，都能均匀染色。文中描述了有助于诊断可能失败原因的光学显微镜标准。低背景染色使得暗视野照明和电视图像分析得以应用。该方法在轴突切断术后仅3至5天的存活时间内有效。因此，使用这种方法和另一种单独描述的方法，可以在取自同一大脑的交替切片中对退化的轴突和轴突终末进行染色，另一种方法在不同条件下可显示退化的轴突终末。
