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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.
Gallyas et al. (1980) studied this question.