Fig. 2

C-terminal TDP-43 peptides elicit high-titer antibodies detecting patient TDP-43 aggregates. TDP-43-specific antibody responses in sera from mice receiving five immunizations (expect TDP1 + 3 with 1–3 immunizations) were analyzed by ELISA, immunofluorescence stainings, and immunoblot. A A dilution series of pooled sera was analyzed by ELISA against recombinant full-length TDP-43 (left panel) or pTDP-43 peptide (pS409/410, right panel). Background-corrected mean OD450 was measured in duplicates. Triangular shapes indicate monogenic animals, circles represent rNLS8 mice (labeling consistent throughout the following figures). B Antibody response in antisera from individual mice was measured as in (A) in single dilutions (1:500 or 1:20,000) to analyze inter-animal variability. Control (ctrl) PBS n = 17, ctrl VAX n = 18, TDP-PBS n = 10, TDP1 + 3 n = 3, TDP5 + 7 n = 13, TDP8 + 9 n = 13, TDP10 + 12 n = 12, and TDP15 n = 12. Note that the number of immunizations in TDP1 + 3 animals (1, 2 or 3) correlates with the resulting titer. C Double immunofluorescence stainings of pooled antisera and phosphorylated TDP-43 (pS409/410) were performed on frontal cortex sections of a sporadic FTLD case. Representative images are shown. Arrowheads indicate strong (TDP10 + 12, TDP15) or weak (TDP1 + 3, TDP8 + 9) labeling of pTDP-43 inclusions with antisera. TDP5 + 7 antiserum failed to detect pTDP-43-positive inclusions (open arrowhead). Images from a healthy control are shown in Fig. S3A. Scale bar = 10 µm. D Immunoblotting of HEK293 cell lysates of doxycycline-inducible TARDBP knockdown (KD) and control (ctrl) using a commercial TDP-43 antibody (left lane) or pooled antisera. Asterisk denotes a prominent non-specific band detected with the ctrl PBS serum. Calnexin was used as a loading control