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3. Acquire an optical image series on the z -axis (i.e., a “ z -stack”)
with images
m apart. The series of images should cover
the whole z -axis range of the brain slice. Preserve the same set-
tings across brain slices on the same slide to ensure similar sig-
nal intensity.
1
μ
1. Open image stacks using ImageJ or similar software. In the
appropriate channel to visualize the nuclear stain, outline and
quantify all the full neuronal nuclei, excluding partial nuclei on
the top and bottom images of the stack. Include only neuronal
nuclei (larger and less-intensely stained than glial cell nuclei).
2. Using the channels appropriate to visualize the both the Alexa
647 (arc) signal and the nuclear counterstain, identify nuclei
with intranuclear arc foci (commonly visualized as one or two
dots within the nucleus). Use a similar procedure to identify
GFP-positive nuclei.
3. Aggregate the cell counts for each animal and calculate the fol-
lowing (a) pArc (Arc+ nuclei/total nuclei in LA), (b) pArc/
GFP+ (Arc + GFP+ nuclei/total GFP+ nuclei), (c) pArc/GFP−
[(total Arc+ nuclei - Arc+GFP+ nuclei)/(Total Nuclei - GFP+
nuclei)]. Figure 3c shows the results from a typical
experiment.
3.4.6 Image Analysis
and Quantifi cation
4
Notes
1. It should be emphasized that amplicon-based HSV viral vectors
are not human pathogens and pose no obvious safety issues.
They cannot replicate and thus have a fi nite lifespan in target
cells. This vector lacks most of the viral genes of wild-type
HSV and integration into the host genome is thought to be
impossible. The helper virus is also replication defective and
unable to enter lytic or latent stages. There may be issues
related to cytotoxicity of contaminating helper viruses in the
generated pool of gene delivery vehicles. However, the band-
ing of the virus on a sucrose gradient followed by a high-speed
centrifugation to pellet the amplicon virus typically is suffi cient
to remove most, if not all, helper virus contamination.
Nevertheless, it remains crucial to exercise caution when
working with these types of replication-defective HSVs and
follow all biosafety regulations. In all procedures, materials
and machinery used for generation of viral particles should be
clearly labeled and used exclusively for virus preparation.
Before and after use, bench tops and other relevant surfaces
should be disinfected with 70 % ethanol or 1 % sodium
hypochlorite, either of which is suffi cient to kill even wild-
type HSV-1. To minimize aerosols, procedures are ideally
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