Biology Reference
In-Depth Information
Blocking solution
0.5 % blocking reagent (Roche #11 096 176 001), 0.1 %
Tween in 1× TBS.
The solution can be stored at −20 °C. On the day of use, thaw
and add serum to 5 % fi nal concentration. Ideally, the
serum should be from the same source of the antibody;
however, we have used normal goat serum generally and
observed no signifi cant background.
TSA-Biotin stock solution
Prepare stock solutions of tyramine (Sigma Cat# T90344,
0.3 M stock solution in DMSO) and biotin-
N
-hydroxy-
succinimide ester (biotin-NHS, Sigma Cat# H1759,
0.3 M stock solution in DMSO). Mix equal volume of the
stock solutions and invert several times gently. The tube is
left rotated gently overnight to mix. The TSA-Biotin solu-
tion can then be stored in −20 °C and stable for up to 3
years.
TSA buffer
In 0.1 M borate buffer:
Adjust with NaOH to pH 8.5
Add 0.01 % H
2
O
2
TSA-Biotin working solution
Dilute TSA-Biotin 1:100 in TSA buffer, add 0.01 % H
2
O
2
Anti-DIG HRP-conjugated antibody (Cat# 200-032-156,
Jackson)
Anti-FITC Alexa488-conjugated antibody (Cat# A11090, Invitrogen)
Alexa647-conjugated Streptavidin (Cat# S-32357, Invitrogen)
Nuclear counterstain (e.g., Hoechst 33258 or DAPI)
Mounting medium for fl uorescence (e.g., Cat# H-1000,
Vectashield)
3
Methods
3.1 Generation
of Herpes Virus
Amplicons for Gene
Delivery
HSV-mediated gene delivery is a commonly used technique in
research laboratories and has been used in our laboratory for sev-
eral years. For delivery and transient expression (7-10 days) of
genes in vivo, a highly effi cient and safe (
see
Note 1
) HSV-amplicon
gene delivery system has been developed by Dr. Rachael Neve and
a detailed protocol has been previously published [
6
,
7
]. HSV-
derived amplicons are highly neurotropic and genetic manipula-
tions of the delivery vectors in this system ensure that they are not
able to replicate within target cells and have negligible chances for
reversion to wild-type virus.
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