Immunohistochemical and Immunocytochemical Detection of Phosphoproteins in Striatal Neurons - Drugs of Abuse: Neurological Reviews and Protocols - page 270

11. Secondary antibody (biotinylated goat anti-rabbit IgG), avidin (reagent A), and
biotinylated horseradish peroxidase (reagent B) are provided by the kit (Vector,
PK-6101).
12. 1 M Tris-HCl stock solution: 121 g of Tris base (TRIZMA base) in 800 mL of
distilled H 2 O. Adjust pH to 7.4 with concentrated HCl (~70 mL of 1 N HCl is
needed to achieve pH 7.4). Adjust volume to 1 L with distilled H 2 O. Store up
to 6 mo at room temperature.
13. Staining solution: 0.25 mg/mL of 3,3 -diaminobenzidine tetrahydrochloride

(DAB, as chromagen to produce reaction products), 0.01% H 2 O 2 and 0.04%
nickel chloride (NiCl 2 ). The heavy metal (nickel) is used to amplify the DAB
reaction product (9) . Dissolve 2.5 mg of DAB; 3.33 L of 30% H 2 O 2 , and 4 mg

of NiCl 2 in 10 mL of 50 m M Tris-HCl, pH 7.4. Shake well and use immediately.
Because DAB is carcinogenic, wear gloves and work in a fume hood.
14. 0.1 M Sodium nitrate.
15. 70%, 80%, 95%, and 100% ethanol and xylenes.
16. DPX mounting medium (Electron Microscopy Sciences).

µ

3. Methods
3.1. Fixation
3.1.1. Transcardial Perfusion for Fixation of Brain Tissue

1. After a lethal dose of anesthesia (chloral hydrate, 500 mg/kg, i.p.), the animal is
moved to a fume hood and an incision on the upper abdomen is made to expose
the diaphragm. Carefully incise the diaphragm to expose the beating heart. To
open the thoracic cavity, two parallel cuts through the rib on either side of the
heart are made, and fold the cut rib fl ap headward with a hemostat. Insert a blunt
15-gauge (rat) or 22-gauge (mouse) hypodermic needle upward from the bottom
apex of the left ventricle through the ventricle and the aortic valve so that the
needle is placed approx 3-5 mm inside the ascending aorta. Clamp the needle in
place with a curved clamp ( see Note 3 ).

2. Perfuse 300 (rat) or 70 (mouse) mL of 4% paraformaldehyde over a period of
5-7 min at 4°C. (Cold paraformaldehyde helps reduce possible blood coagula-
tion during the initial perfusion.) The peristaltic pump is set at 35-40 (rat) or
10 (mouse) mL/min. Immediately after perfusion, cut the right atrium to create
an escape route for the blood and perfusion fl uid. Muscle twitching can be seen if
the fi xative is properly perfused into the animal. After several minutes, stiffening
of the forelimbs and head occurs, a further indication that the fi xative is being
properly perfused ( see Note 3 ).

3. When the perfusion is fi nished, stop the pump and remove the brain from the skull
using a bone rongeur. The brain is postfi xed for 2 h at 4°C in a vial containing
10% sucrose and 4% paraformaldehyde. The brain is then infi ltrated overnight
at 4°C in the cryoprotectant solution until the fl oating brain sinks, indicative of
sucrose infi ltration of the brain. The infi ltration is to prevent freezing artifacts
in the tissue during sectioning.