Rabbit anti cr

For the immunocytochemical detection of CR-positive nerve endings, the extraocular muscle tissue was processed on slides. Sections were pretreated with 10% methanol and 3% H₂O₂ to suppress endogenous peroxidase activity and were then preincubated in 0.1 M PB at pH 7.4 containing 0.3% Triton X-100 with 5% normal goat serum for 1 hour. Then, the tissue was processed with rabbit anti-CR (1:1000; Swant, Marly, Switzerland) for 24 hours at room temperature. The sections were washed in 0.1 M PB three times and treated with biotinylated goat anti-rabbit (1:200; Vector Labs, Burlingame, CA, USA) for 1 hour at room temperature. After three washes in 0.1 M PB, sections were incubated in ExtrAvidin peroxidase (1:1000; Sigma-Aldrich Corp.) for 1 hour at room temperature. Following two rinses in 0.1 M PB and one rinse in 0.05 M Tris buffer solution (TBS, pH 7.6), the antigenic sites were visualized with 0.025% diaminobenzidine (DAB), 0.2% ammonium nickel sulfate (Riedl-De Haën; Germany), and 0.01% H₂O₂ in 0.05 M TBS (pH 7.6) for 5 to 10 minutes, which yielded a black reaction product. The sections were mounted, air dried, dehydrated, and coverslipped in DPX mountant (Merck, Darmstadt, Germany).

Midbrain sections containing the oculomotor nucleus were simultaneously stained for retrogradely transported tracer (CTB or WGA) and CR. The free-floating tissue was washed with 0.05 M TBS (pH 7.6) and preincubated in 0.1 M TBS at pH 7.4 containing 0.3% Triton X-100 with 5% normal donkey serum for 1 hour at room temperature. Then the tissue was processed either with goat anti-WGA (1:250; Vector Laboratories, Burlingame, CA, USA) or with goat anti-CTB (1:5000; List Biological Laboratories) and rabbit anti-CR (1:1000; Swant) in 0.1 M TBS (pH 7.4) containing 0.3% Triton X-100 with 5% normal donkey serum for 24 hours at room temperature. After three washes with 0.1 M TBS (pH 7.4) the sections were treated with a cocktail consisting of Cy³-donkey anti-rabbit (1:200; Dianova, Hamburg, Germany) and Alexa Fluor 488 donkey anti-goat (1:200; Molecular Probes, Eugene, OR, USA) for 1 hour. After three washes with 0.1 M TBS (pH 74) and a short washing in distilled water, the sections were air dried and coverslipped in Fluoromount medium (Sigma-Aldrich Corp.).