Anti dpeaae neo epitope

Transverse frozen sections were cut from the mid-belly of the TA or diaphragm muscle strips at a thickness of 8 μm, mounted on slides and stored at −80 °C until analysis. Immunohistochemistry for ADAMTS1 (Origene, TA317919, Rockville, MD, USA), ADAMTS5, ADAMTS15 (Abcam, ab45047, Cambridge, MA, USA), V0/V1 versican (anti-GAGβ; Merck Millipore, AB1033, Bayswater, VIC, Australia) or versikine (anti-DPEAAE neo-epitope; Thermo Fisher Scientific, PA1-1748A, Scoresby, VIC, Australia) were performed as previously described [9 (link),32 (link)]. An anti-desmin rabbit polyclonal antibody (Abcam, ab15200, Cambridge, MA, USA) together with an anti-rabbit Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific, R37116,) were used to detect myoblasts and newly regenerated myofibres [93 (link),94 (link)]. Representative wild type and mdx TA and diaphragm muscle cross-sections were H and E stained for muscle architecture, and wheat germ agglutinin to assess fibrosis [95 (link)]. For analysis of V0/V1 versican and versikine immunoreactivity, four non-overlapping representative digital images were captured with a confocal microscope of each muscle section at 600× magnification (Olympus Fluoview FV10i) and analysed for area of immunoreactivity using Image-Pro Plus software (Version 7, Media Cybernetics, Silver Spring, MD, USA).

EDL and soleus muscles were homogenized in radio immunoprecipitation assay buffer (RIPA; Merck Millipore) with protease and phosphatase inhibitors (Thermo Fisher Scientific). Total protein content was determined using a BCA protein assay (Thermo Fisher Scientific), and 7.5 µg of unfractionated muscle homogenate was separated on a 4%–15% gradient TGX Stain-Free™ criterion gel (Bio-Rad) at 100 V. Following which the gel was activated and proteins visualized using the Chemidoc™ XRS system (Bio-Rad). Proteins were then transferred to PVDF membranes using a Turbo Blot Transfer System (Bio-Rad) at 2.5 A and 25 V for 12 min. Immunoblotting for versikine (anti-DPEAAE neo-epitope; Thermo Fisher Scientific, PA1-1748A) was performed as previously described [17 (link)]. Blots were imaged using ECL chemiluminescence. Band densitometry was performed on the western blots and the stain free gels, to confirm even loading, using Image Lab software (Bio-Rad). Versikine protein expression was normalized to the optical density of the total protein per lane on the TGX-stain free protein gel.