Using Viral Vectors to Study the Memory Trace in Mice - Viral Vector Approaches in Neurobiology and Brain Diseases - page 141

brain chunks from three to fi ve animals, each from a different
condition ( see Note 8) with the posterior portion facing down.
4. To minimize thawing of the brain, wait for the fi rst layer of
medium to freeze before adding another layer into the
mold. Care should be taken to avoid formation of bubbles,
which will interfere with the sectioning. Repeat this step until
the brain is submerged in the medium. Wait 10-15 min for
the embedding medium to completely freeze, as indicated by a
change from a viscous transparent liquid to an opaque white
solid.
5. Remove the tissue block out of the mold, trim excess medium,
and use embedding medium to secure the brain block to a
specimen disc, with the anterior portion of the brains facing
up. Once the block is frozen to the specimen disc, sectioning
can begin.
6. Use a clean microtome blade to cut the tissue block into 20- m

coronal slices and mount onto microscope slides. To cover the
entire LA, start collecting slices when the dorsal hippocampus
is present and fi nish when the ventral hippocampus starts to
emerge. We typically collect 30-40 slides for each brain block.
Place the slides in a slide box and store at −80 °C where they
are stable for at least 1 year.

μ

1. Linearize the template DNA by enzymatic digestion. For anti-
sense probe, use a restriction enzyme that cuts at 5

3.4.2 In Vitro
Transcription and
Riboprobe Purifi cation

end of the
target gene ( see Note 7). Purify the linearized template (e.g.,
using a commercially available DNA purifi cation column) and
run an aliquot on a 1 % agarose gel to ensure the digestion is
complete.
2. Prepare a 20-

′

l in vitro transcription reaction on ice by mixing
the following: 1

μ

μ g template DNA in RNase-free water (14 μ l),

2 μ l 10× DIG or Fluorescein Labeling Mix, 2 μ l 10× transcrip-

tion buffer, and 2 l RNA polymerase. For antisense probes,

the RNA polymerase used will bind to the appropriate site that
is 3

μ

of the target gene. Incubate the reaction at 37 °C for 1 h.
3. Remove the DNA template by adding 1

′

μ l Turbo DNase I and

incubating at 37 °C for 15 min.
4. Terminate the reaction by adding 2 μ l 0.5 M EDTA (pH 8.0)

l RNase-free dH 2 O. Place the reaction on ice.
5. Purify the riboprobe using an RNA purifi cation column as
described by the manufacturer.
6. Quantify the yield of riboprobe by measuring the absorbance
(A 260 ) using a spectrophotometer. A small amount can also be
taken for denaturing gel electrophoresis alongside RNA molec-
ular weight markers to ensure the probe is the correct size and

and 37 μ