NeuroScience Associates

Disintegrative Degeneration Stain

Applications:

• Neurotoxicity assessment.
• Neuroprotection assessment.
• Identification of “ongoing” degeneration in disease models.
• Identification of intrinsic degeneration (apoptosis) expressed inneonates.

Advantages:

• Capable of detecting disintegration in multiple neuronal elements including cell bodies, axons, dendrites and synaptic terminals.
• Excellent Signal to Noise: Disintegrative degeneration stains black against white/clear background of normal cells.
• Visual detection of results is frequently possible with little or no magnification of brain sections.
• Fewer animals are required for an effective experimental design.

Specifications:

• Survival time planning is crucial. Degeneration persists in a detectable state for ~6 days for this stain (vs. less than 3 days for H&E) from the time degeneration begins.
• NSA’s stain is based on the deOlmos Amino Cupric Silver Stain.
• Use NSA’s perfusion protocols with the paraformaldehyde and phosphate buffer perfusion fix, for optimized staining.
• Use barbiturate anesthetic. Use of gaseous anesthetic tends to prevent flushing of red blood cells which stain with silver, clouding degeneration visibility.

The Disintegrative Degeneration Stain (Amino Cupric Silver Stain) employed by NSA provides the most sensitive and comprehensive detection of neurotoxicity available. This stain is uniquely capable of detecting the death or destruction of neuron cell bodies, dendrites, synaptic terminals and axons. No other class of stain is capable of detecting all of these elements. Other stains typically only detect changes in neuronal cell bodies (e.g. H&E and TUNEL). In addition to the broad range of elements this stain can detect, it represents the definitive example of NSA’s high standard for achieving a high signal to noise image. Degenerating elements are stained black, while normal tissue remains virtually unstained. The extremely effective contrast in this stain necessitates a counterstain (NSA uses neutral red) to stain normal cell bodies for the purpose of anatomical reference.

Experiment Design:

The multiple degenerating elements stained are detectable over a longer period of time than with cell body only stains (i.e. H&E). This creates a broader window of detection which makes it possible to use fewer animals in a study to achieve comparable (actually more comprehensive) results.  To successfully monitor a time period for neurotoxicity, animal groups can be sacrificed at 5-6 day intervals vs. 2-3 day intervals using traditional methods.

Analysis Advantages:

Due to high contrast staining, analysis becomes less time consuming since sections can be visually scanned at a much lower magnification than traditional methods. With proper experiment design, only half the number of sections require analysis due to the benefit of using half the number of animals. Visually scanning tissues with 4X-10X objectives proves sufficient in recognizing the presence of degeneration. A further benefit of the high contrast stain is the potential for digital image analysis. Counts and volumes of degenerating elements are easily detected and calculated by imaging tools and software (see analysis).

For further reference, please see: Stains