Biology Reference
In-Depth Information
(c) Axotomy procedure: At this point, axotomy is usually performed on anes-
thetized animals. The common anesthetics are levamisole (6 mM)
(
Ou et al., 2010
), tetramisole (0.05%) (
Brenner, 1974
), 1-phenoxy-2-pro-
panol (1%) (
Wu et al., 2007
), muscimol (10 mM) (
Hammarlund et al.,
2009
), or simply embedding in high-percentage agar pad (10%). The key
is to make sure that the anesthetics allow rapid immobilization and
recovery of the operated animals, and do not interfere with fluorescent
labeling. The regeneration rate and pattern of axons can be measured by
confocal imaging at different time points of postaxotomy. Advanced
methods using microfluoidics to immobilize animals either by cooling
or pressure have also been reported for axotomy on nonanesthetized
animals (
Guo et al.,2008
), although such methods are not yet widely
used because of technical complications.
E. Questions and Answers
- Are there common methods for quantifying neuronal defects?
Most imaging quantification methods for fluorescent light microscopy can be
used to record expression patterns and levels of fluorescent reporters. For
fluorescence intensity measurement, it is important to maintain identical
image-capture conditions, such as laser power, exposure time, and region of
interest. Several published studies have also devised home-built software
programs for comparing a large number of samples (
Burbea
et al., 2002;
Hung et al., 2007
).
- How does one know if a genetic screen has worked?
A gold standard to determining whether a screen has worked is the frequency of
mutants with behavioral or morphological defects. In the pioneer screen done by
Sydney Brenner, 69 such mutants (uncoordinated, long, dumpy, small, roller, and
blistered mutants) were found in 318 independent F1 plates (
Brenner, 1974
). This
indicates that in a successful screen, at least 20 these mutants can be found in
every 100 F1 plates. Also the percentage of the lethal or sterile mutants can be
used to determine how well a screen has worked. Usually, nearly 30% of F1 plates
will have lethal or sterile mutants.
- How can one reduce false positives in an automated screen?
To minimize false positives in an automatic screen, one needs to set up a good
internal control. The best internal control will be another fluorescent protein,
which is usually RFP if the reporter is GFP, expressed in the adjacent tissue of the
neuron of interest. Also one needs to select a line that has good expression level
for both the reporter internal control. To eliminate false positives, one always
needs to confirm the phenotypes by using compound microscopes before doing
further experiments.
- What should one do when not geting enough cultured neurons?
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