Using Viral Vectors to Study the Memory Trace in Mice - Viral Vector Approaches in Neurobiology and Brain Diseases - page 143

(d) 0.5× SSC—5 min
(e) Twice in 0.5× SSC—30 min each at 56 °C
(f) 0.5× SSC—5 min
(g) 2× SSC—5 min
2. Quench endogenous peroxidase activity by incubating slides in
1 % H 2 O 2 in 2× SSC for 30 min. Cover with tin foil as H 2 O 2 is
sensitive to light.
3. Wash twice in 2× SSC, 5 min each, followed by one 5 min wash
in TBS.
4. Apply 150 l blocking buffer to each slide, coverslip and incu-

bate in humid chamber at room temperature for 30 min ( see
Note 13).
5. Carefully remove coverslips and touch the corner of each slide
on paper towel to remove excess blocking buffer. To each slide,
apply 150

μ

l of HRP-conjugated anti-DIG antibody in block-
ing buffer (1:300 dilution). Coverslip the slides and incubate
in humid chamber for 2 h at room temperature.
6. Remove coverslips and wash three times with TBS-T, 5 min
each.
7. Add 100

μ

l TSA-Biotin working solution to each slide.
Incubate in humid chamber for 30 min at room temperature.
8. Remove coverslips and wash three times in TBS-T, 5 min each.
9. Dilute Alexa 488-conjugated anti-FITC antibody in blocking
buffer (1:100), apply 150

μ

μ l to each slide, coverslip and

incubate at 4 °C overnight.
10. Remove coverslips and wash three times in TBS-T, 5 min each.
11. In TBS, mix Alexa 647-conjugated Streptavidin (1:300) ( see
Note 14) and nuclear counterstain (e.g., Hoechst 33258,
1:1,000). Apply 150 l per slide, coverslip and incubate in

humid chamber at room temperature for 2 h.
12. Remove coverslips and wash three times in TBS-T, 5 min each
13. Wash slides once with TBS for 5 min, apply aqueous mounting
medium and coverslip. The edges can be sealed with nail
polish.

μ

Imaging of hybridization signals for GFP mRNA, Arc mRNA and
the nuclear counterstain is performed using a laser confocal micro-
scope (Fig. 3b ). Serial confocal sections (

3.4.5 Imaging

m) through the LA
are acquired and signals are subsequently quantifi ed using any
suitable image analysis programs. We fi nd that Image-J software
(NIH) works well.

≤ 1 μ

1. Under low magnifi cation, use the nuclear counterstain to
locate the LA. Starting from the dorsolateral edge of the