(A,B) TDP-43 light microscopic images. The pattern of staining suggests nuclear localization and colocalization with DNA staining methyl green confirms this. (C1−3) Colocalization of TDP-43, SMI-31, and DAPI in a single normal muscle section with triple immunofluorescent studies further confirms nuclear localization of both of these proteins. (D1−4) TDP-43 localizes internally to the nuclear membrane as shown in triple immunofluorescent studies with the nuclear membrane protein emerin and DAPI.

(A1-A3) Adjacent sections from an IBM sample show sarcoplasmic areas of intense TDP-43 staining that are not immunoreactive for SMI31 and do not show fluorescence after Congo red staining. (B1-B3) Adjacent sections from an IBM sample show TDP-43 sarcoplasmic staining distinct from SMI31 and DAPI nuclear staining. (C,D) High magnification of TDP-43 immunoreactivity in a myofiber from IBM, with adjacent section showing H&E appearance of this myofiber.

(A-B) TDP-43 sarcoplasmic distribution occurs in non-necrotic myofibers that may have mild abnormalities (enlarged rounded fiber #1; small or angulated fibers #2 and #3) or (C) substantial abnormalities (many slightly basophilic small fibers, some with enlarged nuclei; the vacuolated fiber #4) present on H&E staining.

(A1-A3) Triple stained immunofluorescent section shows vacuoles lined with reactivities for TDP-43, SMI-31, and DAPI. The arrow indicates a myofiber with sarcoplasmic TDP-43, whose nucleus (identified by SMI31 and DAPI staining) does not contain visible TDP-43. (B) Higher magnification of panel A-1, outlining TDP-43 lined vacuoles (arrowheads) and a myofiber with sarcoplasmic non-nuclear TDP-43 (arrow).

(A1−3) A triangular fiber #1 shows abundant sarcoplasmic linear TDP-43 accumulation. Nuclei (marked with arrowheads) are devoid of TDP-43. In contrast, the adjacent rounded myofiber #2 lacks sarcoplasmic accumulation and has normal TDP-43 nuclear immunoreactivity (arrows). Inflammatory cells are present between the two fibers. (B1−3) TDP-43 sometimes clusters around myonuclei (arrowheads) in addition to multifocally within the sarcoplasm. A-1 and B-1 are merged images of TDP-43 (A-2 and B-2) and DAPI (A-3 and B-3) fluorescent signals.

Immunoblots probed with either secondary anti-IgG antibody only (no primary antibody) or antibodies against TDP-43 or actin followed by secondary anti-IgG antibodies. Sample ID for each lane labeled in the bottom panel. An approximately 43 kDa band at the appropriate molecular weight for TDP-43 (black arrow) is seen in all muscle samples and was larger in all IBM samples, despite slightly less actin, than in other inflammatory myopathy samples (DM = dermatomyositis; NM = necrotizing myopathy, PM = polymyositis). The interpretation of the additional band previously reported in IBM muscle30 at approximately 50 kDa (white arrow) is confounded by the presence of abundant immunoglobulin heavy chains present in IBM muscle also at this molecular weight, as shown in the 1^st lane stained only with secondary anti-IgG, without primary anti-TDP-43 antibody. TDP-43 immunoreactivity beyond IgG immunoreactivity may be present at this weight (compare sample 372 without and with primary antibody) but is not disease-specific, as it is present in DM, NM, and PM samples. Immunoreactivity at all other weights also appeared to be not disease-specific, except for 2 IBM samples (354 and 372) that showed greater intensity of multiple lower molecular weight TDP-43 fragments (*).

Comparison of sarcoplasmic TDP-43 abnormalities with other IBM histopathological biomarkers.