Immunohistochemical Localization of SCN

Brain sections (50 µm) containing the SCN were cut on a cryostat at −20°C. Sections were washed 3 times for 10 min with 0.1 M PB containing 0.1% Triton-X-100, blocked for 1 hr with normal donkey serum (NDS) diluted 1∶50 in PB containing 0.3% Triton followed by incubation in the primary antibodies diluted in the same buffer. Pilot studies conducted with primary antibodies at 1∶5,000 dilution revealed that antibodies made in guinea pig generally gave more intense signal than antibodies made in rabbit. Therefore, primary rabbit antibodies were tested at concentrations of 1∶500, 1∶1,000 and 1∶5,000, while guinea pig antibodies were tested at 1∶1,000, 1∶5,000 and 1∶10,000. In cases where these concentrations gave strong background, the guinea pig antibodies were also tested at 1∶20,000 and 1∶40,000. After incubation of primary antibody for 48 hrs at 4°C, sections were washed twice for 10 min, once for 30 min, and once for 10 min in PB+0.1% Triton, and then were incubated for 2 hr in the appropriate secondary antibody (donkey anti-rabbit or donkey anti-guinea pig) conjugated to Cy2 fluorescent chromogen (Jackson ImmunoResearch, West Grove, PA, 1∶200 in PB+0.3% Triton). Sections were washed 3 times for 10 min in PB, mounted, dehydrated and coverslipped with Krystalon (EM Science, Gibbstown, NJ).
In some cases, one of two different amplification protocols was performed. In one, a biotinylated secondary antibody was used (donkey anti-rabbit or anti-guinea pig, 1∶200), followed by incubation in avidin-biotin peroxidase complex (ABC) for 1 hr (ABC Elite kit, Vector Laboratories, Burlingame, CA, USA; 40 µl/10 ml PB+0.3% Triton). In a second amplification protocol (ABC+BT), the biotinylated secondary antibody was followed by incubation in biotinylated tyramine (6 µl/10 ml 0.1 M PB+2 µl H₂O₂ for 30 min). Cy2 avidin (1∶200 in PB+0.3% Triton) was used as the fluorescent label.