1. Fixed tissue was processed as free-floating sections and singly labeled with a polyclonal antiserum raised against choline acetyltransferase (ChAT) from human placenta (1:1000 dilution, Chemicon, CA, USA). The specificity of the antibody has been described extensively (Mesulam et al., 1983; Mufson et al, 1989). 2. Sections were washed several times in Tris-buffered saline (TBS) before incubation with 0.1 M sodium periodate (to inhibit endogenous peroxidase activity) in a TBS solution for 20 minutes.3. After several rinses in a solution containing 0.25% Triton X-100 in TBS, the tissue was placed in a blocking solution containing TBS with 0.25% Triton X-100 and 3% horse serum for 1 hour. 4. Sections were subsequently incubated in goat anti-human ChAT for 48 hours in a solution containing TBS, 1% Triton X-100 and 1% horse normal serum. 5. Following washes with 1% horse normal serum in TBS, sections were incubated with biotinylated horse anti-goat secondary antibodies (Vector Laboratories, CA, USA) for 1 hour.6. After several washes in TBS the tissue was incubated for 60 minutes with an avidin-biotin complex (1:500; Elite Kit, Vector Laboratories, CA, USA). 7. Tissue was rinsed in 0.2 M sodium acetate, 1.0 M imidazol buffer (pH 7.4), and developed in an acetate-imidazol buffer containing 2.5% nickel sulfate, 0.05% 3,3-diaminobenzidine tetrahydrochochloride (DAB; Sigma-Aldrich, St. Louis, MO, USA) and 0.0015% H2O2. The reaction was terminated using an acetate-imidazol buffer solution. 8. Finally, sections were mounted on glass slides, dehydrated in graded alcohols, cleared in xylenes and cover slipped with DPX (Biochemica Fluka, Switzerland). All sections were processed at the same time using the same chemical reagents to avoid batch-to-batch variation during immunostaining.