Figure 8. .

Characterization of R416W GFAP-specific antibodies and demonstration of its presence in Rosenthal fibers. A, Immunoblots, performed as described in the “Material and Methods” section, with use of purified, recombinant human wild-type (hGF-WT) and R416W (hGF-R416W) GFAP and lysates from brain samples taken from either control human (WT) or patients with Alexander disease that harbor either an R239C mutation (R239C) or an R416W mutation (R416W) in GFAP. The general anti-human GFAP monoclonal antibody (SMI-21) reacts with all samples, whereas the anti-R416W monoclonal antibodies (1A3 and 19.2) produce signals from R416W-containing samples only, with the pattern for the R416W patient lysate identical to that of SMI-21. The identity of the immunopositive bands above and below the prominent GFAP-positive band are as yet unknown. The lower bands most likely correspond to degradation products, since these are normally seen in control brain samples. The upper bands are common to both the R239C and R416W samples, suggesting these are a common feature of Alexander disease pathology, but, as yet, the reason for their slower electrophoretic mobility is unknown. Panels B and C are striatum in a control (normal) brain stained with standard polyclonal GFAP antibody (B) and by R416W monoclonal antibody 19.2 (C). Note that the R416W antibody does not crossreact with normal human brain tissue. Panels D–F are brain sections from a patient with the R416W GFAP mutation that are stained with the monoclonal 19.2 antibody and then are visualized by either peroxidase- (D) or rhodamine-tagged secondary antibodies (F) or are stained with the rabbit polyclonal GFAP antibody (Dako) and then are detected with FITC-tagged secondary antibodies (E). Nuclei are counterstained with Hoechst 33258 (E and F [see the “Material and Methods” section for procedure details]) to assist comparison of the panels E and F. Numerous Rosenthal fibers are stained around their periphery (arrows in E and F), a feature often reported for these aggregates (e.g., the work of Tomokane et al.⁶). Normal-looking GFAP filaments are also stained by the R416W-specific mAb (arrowheads in F) and can be detected by the diffuse staining in other parts of the section.