Using Viral Vectors to Study the Memory Trace in Mice - Viral Vector Approaches in Neurobiology and Brain Diseases - page 142

that is has not undergone degradation ( see Note 9). The
remainder may be aliquoted and stored at −80 °C until needed.

All the steps in this section should be performed under RNase-free
conditions ( see Note 10). It is also advisable to perform the experi-
ment in a designated RNase-free area.

3.4.3 Hybridization

1. Choose slides evenly spaced over a wide range of the anterior-
posterior axis of the entire LA. Thaw the slides on a fi lter paper
and use a pencil to mark the slides. Do not use permanent
markers as the marks will be washed away with acetone/meth-
anol treatment.
2. Load slides onto autoclaved metal rack and transfer through
the following solutions (250 ml) in glass jars:

(a) Ice-cold 4 % PFA—10 min

(b) 2× SSC—2 min
(c) Acetic anhydride (made immediately before use)—10 min
(d) Ice-cold acetone/methanol—5 min
(e) 2× SSC—5 min
3. Apply 200 l of prehybridization buffer to each slide and cov-

erslip. Incubate in a humid chamber for 30 min at room tem-
perature ( see Note 11).
4. Prepare hybridization solution containing the riboprobes. Add
FITC-conjugated GFP antisense probe and DIG-conjugated
arc antisense probe to 1× hybridization buffer (fi nal concentra-
tion of 0.67 ng/μl

μ

μ l for each probe). Heat at 90 °C for 5 min

and place on ice.
5. Apply 150 l hybridization buffer per slide, coverslip and place

the slide in the humid chamber. Incubate the slides overnight
at 56 °C.

μ

These steps do not need to be performed under RNase-free condi-
tions. Brain slices are fi rst washed to remove unhybridized probes,
followed by quenching of endogenous peroxide activity. Arc ribo-
probe is detected by incubating with HRP-conjugated antibody
followed by tyramide amplifi cation and treatment with Alexa-647
conjugated streptavidin ( see Note 12). The GFP riboprobe is visu-
alized using an Alexa-488-conjugated FITC antibody.

3.4.4 Washing and
Fluorescent Staining

1. Remove the coverslips by dipping the slides in 2× SSC and
then place the slides in a metal rack for the following washes/
treatments at room temperature (unless otherwise indicated):

(a) Twice in 2× SSC—10 min each

(b) 2× SSC with 1 ng/ml RNase A—30 min at 37 °C

(c) Twice in 2× SSC—10 min each