Double immunofluorescence analysis and Western blot analysis of TDP-43 PFFs M1-C mice at 4 mpi. a1–e3 Representative images of immunofluorescence were analyzed to demonstrate the aggregation of pathological TDP-43 in different types of cells. Double immunofluorescence analysis of cortex, pons, medulla oblongata and cervical spinal cord from TDP-43 PFFs M1-C mice for pTDP-43 (red, a2-e2) with MAP2 in M1(green, a1) and parvicellular reticular nucleus (green, b1), ubiquitin in cervical spinal cord (green, c1), GFAP in LGP (green, d1), Iba-1 in MdD (green, e1). f1–g3 Double immunolabeling for MBP (green) and neurofilament (red) in dorsal corticospinal tract in the ipsilateral side (f1–f3) and contralateral side (g1–g3) of injection from TDP-43 PFFs M1-C mice. Co-immunolabeling is represented by signal in yellow. Cell nuclei were counterstained with Hoechst 33258 (blue). [Scale bars, 30 μm (a1–g3)]. WB analysis of pTDP-43 in the soluble and insoluble fractions from cortex (h), red nucleus (j), decussatio pyramidum (l) and cervical spinal cord anterior horn (n) using the anti-pTDP-43 (phosphorylated at Ser409/Ser410) antibody. Blots were probed for GAPDH as a loading control (Bottom). Molecular weight markers of migrated protein standards are expressed in KDa. Quantification of soluble and insoluble pTDP-43 levels in cortex (i), red nucleus (k), decussatio pyramidum (m) and cervical spinal cord anterior horn (o) (n = 3 mice/group) show plentiful pTDP-43 in the extracts of TDP-43 PFFs M1-C mice while few in age-matched PBS M1-C mice. The error bar in panels (i, k, m, o) represents the Standard Error of Mean (SEM). Data are the mean ± SEM. Statistical significance was analyzed using the Student’s t test and Mann–Whitney test, ***P < 0.001.