Summary of patient and control muscle biopsies.

<p><i>Tibialis anterior</i> (TA), <i>tibialis posterior</i> (TP), <i>gastrocnemius lateralis</i> (GL), <i>gastrocnemius medialis</i> (GM), <i>soleus</i> (SOL), <i>extensor hallucis longus</i> (EHL), <i>extensor digitorum longus</i> (EDL), thigh posterior (THP), immunofluorescence microscopy (IF), western blotting (WB) and immunohistochemistry (IHC). The expression array controls (C1, C2 & C5) and (C3 & C4) were collected from amputation material from the distal lower limb muscles of two individuals.</p

Immunofluorescent microscopy of TMD and control biopsies.

<p>(A) HSPA5 staining showed the presence of cytoplasmic granular dots in non-vacuolated TMD muscle fibres. The LC3B channel showed the area has no vacuolated fibres and no autofluorescent material present. (B) A control of HSPA5/LC3B double staining was included for comparison. (C) LAMP2 and LC3B double staining of a TMD muscle fibre showed a representative rimmed vacuole with massive accumulation of LC3B inside and negligible signal from LAMP2 around the edge of the fibre. The top right corner of the LC3B image showed a rimmed vacuole in bright field. (D) A control of LAMP2/LC3B double staining was included for comparison. All images are nuclear counterstained with DAPI (blue signal). Sections are from samples TMD 6 and Ctrl 3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090819#pone-0090819-t001" target="_blank">Table 1</a>. Scale bar 50 µm.</p

Histology and immunohistochemical analysis of TMD muscle biopsies.

<p>(A) Herovici histology staining of TMD muscle sections showing rimmed vacuolar regions within the fibres. (B) Immunohistochemical (IHC) analysis of TMD muscle sections stained for ubiquitin showing localised cytoplasmic ubiquitin containing inclusions in atrophic and rimmed vacuolated fibres and immunoreactivity at the edge of rimmed vacuoles. (C) Autophagosome marker LC3B staining showed strong immunoreactivity within the rimmed vacuolar regions. (D) VCP staining showed immunoreactivity in bodies within a large subset of rimmed vacuoles. (E) p62 staining showed immunoreactivity in rimmed vacuoles and granular immunoreactive dots (shown by an arrow) in an atrophic fibre. (F) Lysosomal protein LAMP2 staining showed no overall increase in rimmed vacuolated fibres. Two separate TMD fibres (A–C and D–F) with rimmed vacuoles in serial sections are shown. Controls of (G) Herovici staining, (H) ubiquitin, (I) LC3B, (J) VCP, (K) p62 and (L) LAMP2 IHC are included for comparison. Sections are from samples TMD 6 and Ctrl 3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090819#pone-0090819-t001" target="_blank">Table 1</a>. Scale bar 50 µm.</p