To begin to characterize the types of retinal cell that express semaphorins, retinal sections from P14 rats that had been hybridized for semaphorin-3B were subsequently immunostained with a variety of antibodies. After in situ hybridization, the sections were stored in PBS overnight at 4°C. All antibodies were diluted in a solution consisting of 1% BSA, 0.2% Triton X-100 in PBS. Antibodies used were raised against: neuronal class β-III tubulin (clone TUJ1, 1:500 dilution; Covance, Vienna, VA), calbindin D-28K (1:1000 dilution; Sigma-Aldrich, Castle Hill, Australia), parvalbumin (PV-28, 1:1000 dilution; Swant Immunochemicals, Bellinzona, Switzerland), or protein kinase Cα (PKCα, 1:300 dilution; BD Transduction Laboratories, Lexington, KY). Some P14 retinal sections hybridized for semaphorin-3A and some adult retinal sections hybridized for semaphorin-3B were also immunostained for β-III tubulin. Sections were incubated with primary antibodies overnight at 4°C and washed three times for 5 minutes each with PBS. Secondary antibodies were then applied (anti-mouse FITC, 1:100; ICN, Aurora, OH; or anti-rabbit FITC, 1:200; Sigma-Aldrich) and incubated for 2 hours at room temperature. Sections were again washed three times for 5 minutes each with PBS before they were coverslipped (Citifluor; Agar Scientific, Ltd., Stansted, UK).