Mice were deeply anesthetized and perfused transcardially with 0.1 M PBS (pH
7.4) containing 0.1% heparin, followed by 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4). The brains were removed, postfixed overnight, washed in
phosphate buffer at 4°C, and cryoprotected in phosphate buffer containing
30% sucrose. Free floating sections at 20 or 40 µm were generated on a
freezing microtome. Primary antibodies used were anti-p11 (1:200; goat antibody;
R&D Systems), anti-EGFP (ab290, 1:10,000; Abcam, Inc., Cambridge, MA), and
anti-c-Fos (K-25, 1:1000; Santa Cruz Biotech) anti-RFP (1:1000; rabbit antibody,
Rockland-inc), anti-CRE (2D8, 1:1000, mouse antibody, Millipore), anti-substance-P (1:400,
rabbit antibody; Millipore) and anti-Met-enkepahlin (1:250, rabbit antibody, Abcam, Inc.).
Secondary biotinilated antibodies, Vectastain Elite ABC kit, Mouse on Mouse kit and
Avidin-Streptavidin blocking reagents were from Vector Laboratories. Blue DAB reagent
contained 0.05% DAB, 0.05% Cobalt Chloride, 0.05% Nickel Ammonium
Sulfate and 0.015% hydrogen peroxide. Brown DAB reagent contains 0.03% DAB
and 0.015% hydrogen peroxide. Fluorescent secondary antibodies (Alexa-conjugated
donkey anti-rabbit and donkey anti-mouse) were from Invitrogen.
7.4) containing 0.1% heparin, followed by 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4). The brains were removed, postfixed overnight, washed in
phosphate buffer at 4°C, and cryoprotected in phosphate buffer containing
30% sucrose. Free floating sections at 20 or 40 µm were generated on a
freezing microtome. Primary antibodies used were anti-p11 (1:200; goat antibody;
R&D Systems), anti-EGFP (ab290, 1:10,000; Abcam, Inc., Cambridge, MA), and
anti-c-Fos (K-25, 1:1000; Santa Cruz Biotech) anti-RFP (1:1000; rabbit antibody,
Rockland-inc), anti-CRE (2D8, 1:1000, mouse antibody, Millipore), anti-substance-P (1:400,
rabbit antibody; Millipore) and anti-Met-enkepahlin (1:250, rabbit antibody, Abcam, Inc.).
Secondary biotinilated antibodies, Vectastain Elite ABC kit, Mouse on Mouse kit and
Avidin-Streptavidin blocking reagents were from Vector Laboratories. Blue DAB reagent
contained 0.05% DAB, 0.05% Cobalt Chloride, 0.05% Nickel Ammonium
Sulfate and 0.015% hydrogen peroxide. Brown DAB reagent contains 0.03% DAB
and 0.015% hydrogen peroxide. Fluorescent secondary antibodies (Alexa-conjugated
donkey anti-rabbit and donkey anti-mouse) were from Invitrogen.