Re: Silver Degeneration Stain for CNS

From:"J. A. Kiernan"

On Mon, 12 Nov 2001, Jen, Shirley {Path~Palo Alto} wrote:

> Does anyone know if there is any silver impregnation staining technique that
> works on paraffin section to stain degenerated neuronal cells?  Or has
> anybody have an experience in doing de Olmos' amino-cupric-silver stain and
> does it work on paraffin section fixed in regular 10% formalin, instead of
> perfusion with 4% paraformaldehyde and stained using free-floating frozen
> section?  We would like to avoid de Olmos' technique, if possible.  Your
> expertise is greatly appreciated.

The short answer is, "Probably there isn't one that's any good."
There are 3 types of silver methods for degenerating axons:

1. Ones that also stain normal axons, exemplified by the Glees
   method. One variant (Glees & Marsland) is specifically for
   paraffin sections. I used it years ago; you have to search
   with am oil immersion objective for the degenerating synaptic
   terminals, and false-positives occur in some normal parts
   of the brain, including the hypothalamus.
2. Ones with a step to reduce staining of normal fibres. The
   Nauta-Gygax method shows degenerating axons of passage and
   pre-terminal parts of axons, but not the synaptic terminals
   themselves. The Fink-Heimer method shows the terminals, and
   was the most popular technique before the 1970s when degeneration
   methods were replaced by tracing methods based on axonal
   transport. I don't know of a variant for paraffin sections.
   If there is one, it might not work well because there's
   evidence from histochemical experiments that lipids are at
   least partly responsible for the stainability of the
   degenerating axons. 

For references and discussion, see Chapter 1: Neuroanatomical 
Methods, in "Methods in Brain Research," ed. P. B. Bradley. 
London: Wiley, 1975, pp 1-77.

These methods are all quite difficult to do, and prone to
inexplicable failures, dirty black messes everywhere etc.
The survival time after the lesion is critical (optimum
4 days for Glees, 7 days for Nauta, but variable and needs
to be determined for each system being investigated, The
specimens must be adequatelly fixed: typically a week or
two in a formaldehyde solution. Thick sections (30-80 um) 
are desirable for any kind of axonal tracing.

The De Olmos technique (De Olmos JS,  Beltramino CA & De 
Lorenzo SDO 1994. Neurotoxicology & Teratology 16: 545-561) 
was introduced for showing somata and larger dendrites of
injured neurons, as soon as 15 minutes after local excitotoxic
injury and also after retrograde transport of doxorubicin, a
toxic antibiotic. The uses of this method and Fink-Heimer
have been reviewed recently by Switzer RC 2000. Applicability 
of silver degeneration stains for neurotoxicity testing. 
Toxicologic Pathology 28: 70-83.

John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1

<< Previous Message | Next Message >>