Using Viral Vectors to Study the Memory Trace in Mice - Viral Vector Approaches in Neurobiology and Brain Diseases - page 147

DEPC-treated dH 2 O, when possible. In addition, it is advisable
to use a designated set of pipettes along with fi lter tips for
working with RNA.
10. The paraformaldehyde fi xes the tissue mildly, preserving RNA
signals while also allowing probe penetration into the cell.
Acetic anhydride treatment acetylates protein side chains and
reduces background, while also have an effect of inhibiting
RNases, resulting in better signal. Acetone/methanol treat-
ment increases cell membrane permeability.
11. A simple way of constructing a humid incubation chamber
involves soaking a piece of fi lter paper with a solution of 50 %
formamide/2× SSC and placing it at the bottom of the cham-
ber tray (not touching the slides).
12. Streptavidin forms a tetramer upon binding to a single biotin
molecule. Therefore, incubation using TSA-biotin followed
by Alexa647 conjugated streptavidin treatment can provide a
further amplifi cation of the arc signal.
13. For this step, the humid chamber is created by soaking fi lter
paper in TBS.
14. Alternatively, using Alexa 568 conjugated streptavidin will
yield a stronger signal than its Alexa 647 counterpart and pro-
vide additional advantage of easier visualization of arc through
a low magnifi cation ocular lens. However, since GFP signal is
much stronger than that of arc, we have observed occasional
“bleeding over” of GFP signal into adjacent Alexa 568 chan-
nel, resulting in false GFP/arc double positive cells during
image acquisition. Using Alexa 647 minimizes this potential
problem.
15. Typically, sections that have more than 30 GFP+ cells are
included for analysis.
16. We have occasionally observed auto-fl uorescent particles or
dots in the background which can interfere with counting arc
signal. The control red channel can be used to identify auto-
fl uorescence, the signal of which can then be subtracted to
reveal the true arc signal.

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