CNS and PNS tissues were fixed by transcardial perfusion with 4% paraformaldehyde in 0.1 M sodium phosphate, pH 7.4. After extensive washing with 0.1 M sodium phosphate, pH 7.4, the tissue was cryoprotected by immersion for 15 min each in 5%, then 10% sucrose in 0.1 M phosphate buffer, pH 7.4, and finally overnight in 25% sucrose in 0.1 M phosphate buffer, pH 7.4, at 4°C. After embedding in OCT (Tissue TEK) blocks were frozen in isopentane cooled in liquid nitrogen. Sections (8–10 μm) were collected on 3-aminopropyltriethoxysilane subbed glass slides, and OCT was removed by washing in PBS (Sigma Chemical Co.). Sciatic nerve fibers were teased in 0.1% Triton X-100 in PBS. Before incubation with the NFF3 antibody, sections or teased sciatic nerve fibers were treated with Bouin's reagent for 1 min. Samples were blocked for 3 h in 0.2% Triton X-100, 0.2% gelatin in PBS, pH 7.4, containing either 10% nonimmune goat serum when using rabbit and mouse primary antibodies, or donkey serum when the sheep antibody was used. The antibodies were applied overnight in 4% nonimmune serum in the same buffer, and after washing them in buffer without serum, fluorescently labeled secondary antibodies were applied for 2 h in buffer containing serum, followed by further washes. Secondary antibodies were as follows: FITC-labeled goat anti–rabbit (Cappel Laboratories), TRITC-labeled goat anti–mouse IgG1, TRITC-labeled donkey anti–rabbit (Jackson Laboratories), and FITC-labeled donkey anti–sheep (Jackson Laboratories). Further washes in blocking buffer minus serum were followed by several washes in PBS. Sections were mounted in Vectashield (Vector Laboratories). Images were captured with a Leica TCS4D confocal or Olympus BX60 microscope, and figures were produced with Adobe Photoshop.