**6.4 Immunohistochemistry**

Fixed, 1-mm transversal slices of optic lobes were included in Paraplast (Oxford Labwase, St. Louis, MO, USA) following the manufacturer's instructions. Microtome slices of 10 μm were cut, transferred to glass slides, de-parafinized, and rehydrated by standard procedures. Slices were incubated in PBS pH 7.4 containing 0.1 M glycine for 30 min at 4°C to block aldehyde groups and then washed three times for 10 min in PBS. They were then incubated in the dark at room temperature for 30 min in methanol containing 0.9% hydrogen peroxide solution to inhibit endogenous peroxidase activity, followed by washing in PBS. The samples were permeabilized and blocked by incubation in PBS containing 1% Triton X-100, 3% BSA, and 0.5% sheep serum and then incubated for 1 h with primary antibodies in PBS containing 0.1% Triton X-100, 3% BSA, and 0.5% sheep serum. After washing in PBS containing 0.1% Triton X-100, the slices were incubated for 1 h with secondary antibodies conjugated to horse radish peroxidase (KPL, Gaithersburg, MD, USA) diluted 1:400 in PBS containing 0.1% Triton X-100, 3% BSA, and 0.5% sheep serum and developed using 3,3-diaminobenzidina (DAB) as substrate.

### **6.5 Immunofluorescence**

Synaptosomes in suspension were fixed in 1% paraformaldehyde for 6 hr. at room temperature, adhered to glass microscope slides by incubation for 1 hr. After gentle washing with PBS, slides were incubated for 30 min in 0.1 M glycine, 0.3% Triton X-100 in PBS, pH 7.4, and washed and blocked in 1 mg/ml BSA, 1% goat serum, and 1% Triton X-100 for 1 hr. at room temperature. The slides were washed with PBS containing 0.3% Triton X-100 and incubated with primary antibody in PBS containing 1 mg/ml BSA, 1% goat serum, and 0.3% Triton X-100 for 2 hrs. at room temperature. The slides were washed in PBS and incubated with appropriate secondary antibody for 1 hr. and washed again. The slides were mounted in Fluoromount G (EMS) diluted 2:1 in PBS and examined on a Zeiss 510 confocal microscope.
