Figure 6.

Distribution of neurite lengths of tectal cells in the presence and absence of antibodies to CALEB. Culture dishes were coated with immunoaffinity purified F11 (A and D), NgCAM (B, E, and F), or laminin-1 (C) and blocked with culture medium containing FCS. Dissociated tectal cells were cultivated on NgCAM or laminin-1 (LN) for 22 h and on F11 for 30 h in culture medium containing FCS. Control cultures were grown without antibodies and test cultures in the presence of Fab fragments of antiCALEB antibodies from four different rabbits (Nos. 1, 4, 6, or 7) using different concentrations as indicated in each panel (300–1,000 μg/ ml). Purified mAb 4/1 was used at 10 μg/ml. In D, tectal cultures were grown on F11 without antibodies, in the presence of Fab fragments of antibodies to CALEB, or in the presence of Fab fragments preincubated with immunoaffinity-purified CALEB immobilized on nitrocellulose (neutralized; for details see Materials and Methods). In E, tectal cells were grown on NgCAM in the absence of Fab fragments or in the presence of both Fab fragments (800 μg/ml) and immunopurified CALEB (10 μg/ml). Cultures were fixed, stained, and analyzed using an inverted microscope and an image analysis system. The percentage of neurons (vertical axis) with neurites longer than 20 μm (horizontal axis) is plotted as introduced by Chang et al. (1987). For each experimental condition, 90–100 neurites were measured. The broken lines in A, B, C, and D indicate that there are no neurons with neurites at the indicated Fab concentration. (The length distributions have been analyzed using the Mann-Whitney U-test and relevant p-values are given: in B, control versus Fab at 330 μg/ml P < 0.0001; in C, control versus Fab at 400 μg/ml P < 0.0001; in E, control versus Fab plus CALEB P < 0.01.)