Additional file 6 of The double homeodomain protein DUX4c is associated with regenerating muscle fibers and RNA-binding proteins

Additional file 6: Figure S5. DUX4c detection by immunohistochemistry in muscle sections and histological alterations in FSHD muscles. (A) Immunostaining was performed with the rabbit serum raised against a DUX4c peptide (2) and secondary antibodies coupled to HRP on healthy muscle sections using the TSA amplification system with DAB detection. PAS counterstaining (pink) delimits muscle fibers, including satellite cells. Pictures were only taken in areas presenting DUX4c-positive nuclei (black arrows). Yellow arrows point to DUX4c-negative nuclei. (B-C) DUX4c immunostaining was performed on FSHD muscle sections as in (A) except a standard procedure was used with DAB detection and hemalun counterstaining (negative nuclei in blue, yellow arrows). The black arrows indicate strong DUX4c labeling in myonuclei at the periphery of fibers adjacent to an angular fiber (star, left panel) or presenting delocalized nuclei (right panel). Yellow arrows highlight DUX4c-negative nuclei. DUX4c staining could also present a granular aspect in the sarcoplasm (star) or extend from a peripheral nucleus to just under the basement or the sarcoplasmic membrane (arrowheads). (C) Sections of FSHD muscles from a single patient were treated (as in Fig. 2C) in parallel to detect DUX4c: either no (infraspinatus, IS, muscle) or variable DUX4c immunostaining from scarce (sub-scapularis, SS, muscle: in some nuclei at the periphery of a degenerating fiber) to several positive peripheral nuclei (serratus posterior inferior, SPI, and intercostalis, I, muscles). (A-C) Negative controls (boxed panel) correspond to either omission of the primary antibody or antigenic peptide competition on consecutive sections. (D) Muscle sections derived from patients presenting a CSS <5 (upper panels) or > 5 (middle and bottom panels) (characterized in Table S4) were stained with Heidenhain blue trichrome. The bottom panels present a cluster of hypotrophic myofibers with peripheral or delocalized nuclei (red arrowheads) and myofibril disorganization detected as branched or split sarcomeres (square) or presenting partial loss of sarcomeric regions (arrowhead). A degenerated muscle myofiber (in the center of the image) is characterized by acidophilic myofibrils (dark pink), vacuolar sarcoplasm and pyknotic delocalized nuclei. Further degeneration is shown by the presence of ghost fibers only detected by their empty basal lamina (PAS diastase stain).