Goat anti wga

Free-floating immunohistochemistry was done after pretreatment (1% peroxide in ice cold methanol for 20 min, 0.3% glycine in phosphate buffered saline (PBS) for 10 min and 0.03% SDS in PBS for 10 min) and blocking (3% normal donkey serum in PBS/0.25% Triton X-100 for 1–3 h). All primary and secondary antibodies were diluted in blocking solutions. Primary antibodies were incubated at 4 °C at least over night or for 48 h and secondary antibodies were incubated at room temperature for 2 h. For rabbit anti-pSTAT3 (1:1000, Cell Signaling) and rabbit anti-cFos (1:5000, Millipore/Calbiochem) staining was developed with ABC/3,3′ diaminobenzidine tetrahydrochloride (Pierce DAB Substrate Kit, ThermoScientific) as described earlier [69] (link). Double labeling was done with either DsRed (1:1000, rabbit anti-DsRed, Santa Cruz), WGA (1:1000, goat anti-WGA, Vector Laboratories), GFP (1:000, chicken anti-GFP; AbCAM), or orexin/hypocretin-A (1:1000, goat anti-orexin/hypocretin-A, Santa Cruz) was visualized with fluorophor-labeled secondary antibodies (1:200 Alexa 568 or 488, Invitorgen or 1:400 Dylite, Jackson ImmunoResearch). Sections were mounted onto gelatin-subbed slides and cover slipped with ProLong Anti-fade mounting medium (Invitrogen) for further microscopy analysis.

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.).