Biotinylated anti rabbit igg secondary antibody

Immunoelectron microscopy was carried out as described previously with minor modifications.^{35 (link),36 (link)} Eyes were enucleated and fixed in 4% paraformaldehyde in PBS buffer (10 mM sodium phosphate, pH 7.5, 0.9% saline) for 3 hours, and the anterior part of the eye was removed. After four washes in PBS, the retinas were embedded in buffered 7% agarose TypeX1 (Sigma-Aldrich Corp., St. Louis, MO, USA). Agarose blocks were sectioned with a vibratome (Leica, Wetzlar, Germany) in 100-μm slices. vibratome sections were blocked in 20% normal goat serum in PBS overnight at 4°C and subsequently incubated with anti-rabbit antibody against RS1 in PBS plus 5% goat serum for 2 days at 4°C. After washing, the sections were incubated with the biotinylated anti-rabbit IgG secondary antibody (Vector Laboratories, Inc., Burlingame, CA, USA) for 4 hours at RT. Then the sections were incubated with a complex consisting of avidin and biotinylated horseradish peroxidase (Vectastain ABC-Kit; Vector Laboratories) for 2 hours at RT in the dark. Subsequently, the sections were incubated in peroxidase substrate solution to obtain the desired staining intensity. The stained sections were then postfixed in 2.5% glutaraldehyde and 1% osmium, respectively; after dehydration, they were embedded in epon. Ultrathin sections were examined in a JEOL JEM-2100 transmission electron microscope (Tokyo, Japan).