|
Introduction |
Scope of Stained Elements |
Compare With H&E |
Silver Degeneration Staining
Evolution |
Neurotoxicity Detection Paper
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Applications:
-
Neurotoxicity
Assessment.
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Neuroprotection
Assessment.
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Identification of
"ongoing" degeneration in disease models.
-
Identification of
intrinsic degeneration (apoptosis) expressed in neonates.
Right images: Massive degeneration in
Soman treated rat. Amino Cupric Silver with neutral red counterstain |
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.
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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
Na Cacodylate 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. (See:
“Neurotoxicity Detection” Paper) 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 (see
NEUROTOXICITY DETECTION paper). 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
offered by NSA Labs). |
