Viral-Mediated Gene Transfer to Study the Behavioral Correlates of CREB Function in the Nucleus Accumbens of Rats - Drugs of Abuse: Neurological Reviews and Protocols - page 336

3.6. Immunohistochemistry
The following are details specifi c to our immunohistochemistry procedures
for CREB (or mCREB, expression of which is indistinguishable from that of
CREB using the antibodies described here):

1. Section the brains on a freezing microtome at 20 m. We use 18-well containers

fi lled with 0.005% sodium azide. For each brain we use six wells, such that each
well contains every sixth section. We use at least one well of tissue for Nissl stain-
ing, whereas the other fi ve wells can be used for various immunohistochemistry
analyses (replicates with one antibody, analyses with several different antibodies).

µ

2. We use a blocking buffer containing 2% NGS and 1% BSA, to maximize
suppression of background CREB expression.

3. We incubate overnight at 4°C in rabbit polyclonal antibody to CREB (1 1000)

within the NGS-BSA blocking solution.

4. We incubate for 2 h at room temperature in goat biotinylated anti-rabbit IgG
(1 200).

5. We mix the avidin-biotin complex at least 30 min before incubating the sections
in it for 1 h at room temperature.

3.7. Behavioral Assessment
The most critical element in our place conditioning procedures is the use of
an “unbiased” place conditioning apparatus. An unbiased apparatus is one in
which the rats have no reliable a priori preference for any of the compartments,
although it is important that each compartment have qualities that allow the
animals to discriminate among environments. Our apparatus has three types
of distinguishing features: wall color (white and black), fl oor texture (rods
and screen), and most importantly, light intensity (dim to bright) ( Fig. 4 ;
see Note 7 ).

1. Place the apparatus in a quiet room. Minimize personnel entry into the room
during place conditioning sessions, because disruptions may change the behavior
of the rats. All portions of the place conditioning procedure should be conducted
during the light portion of the activity cycle with the lights off in the test room.
These conditions maximize exploratory behavior in the apparatus.

2. Before each screening session, ensure that all chambers in the apparatus are
accessible (i.e., doors open) and clean. To minimize olfactory cues associated
with other rats, clean the entire apparatus with isopropyl alcohol wipes if
necessary ( see Note 8 ). Leave the tops open until ready for testing to minimize
trapping odors within the apparatus.

3. Day 0 . Close the tops of the side compartments. Gently place a naive rat into
the center compartment of each apparatus, and close the top. Turn the lights
off in the test room.