Ncl hamlet

To visualize dysferlin expression, we isolated and fixed TA and quadriceps muscle with acetone in −20°C for 30 min; each muscle was sectioned by 10-μm thickness. The sample was washed with PBS, and nonspecific binding sites were blocked with 3% BSA in PBS for 30 min. The fixed muscle was incubated with specific dysferlin antibody (NCL-Hamlet; Leica Biosystems,Lincolnshire, IL, USA) for overnight. Muscle was washed with PBS and then incubated with fluorescein isothiocyanate (FITC)-conjugated IgG (Invitrogen, San Jose, CA, USA). For IgG staining, muscle section was incubated with Alexa Fluor 488 anti-mouse IgG (Invitrogen, San Jose, CA, USA) antibody in 3% BSA for 2 h. After washing muscle sections with PBS, the stained muscle was mounted and visualized by immunofluorescent microscope (Eclipse 80i; Nikon Corporation, Japan).

Immunohistochemical analysis for dysferlin in muscle samples was performed in the institution or hospital that the patient visited. Although the detailed methods may not have been identical in all cases, most cases were investigated by the avidin–biotin–peroxidase complex immunostaining method using the mouse monoclonal antibody to dysferlin (NCL-Hamlet; Leica Biosystems, Wetzlar, Germany) as the primary antibody.

Proteins were extracted from differentiated iFDM cells using Radio-Immunoprecipitation Assay (RIPA) buffer (40 mmol/L Tris–HCl pH 8, 150 mmol/L sodium chloride 1% Triton X-100, 0.5% sodium deoxycholate, 0.5% SDS) with protease inhibitors (complete, Roche, Indianapolis, IN, USA) then a sample was quantified using a BCA (bicinchoninic acid) protein assay (Thermo Fisher Scientific, Waltham, MA, USA). The remaining protein was heated at 70°C in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Laemmli sample buffer 10 min then separated on 7.5% acrylamide TGX SDS–PAGE gels (Bio-Rad, Hercules, CA, USA), blotted onto nitrocellulose filters using an iBlot Gel Transfer Device (Life Technologies, Grand Island, NY, USA, program P3, 10 min), and analyzed by western immunostaining using LI-COR Odyssey blocking reagent and methods as described.30 (link) Primary antibodies included and anti-DYSF specific for the C-terminal end (NCL-Hamlet, Leica Biosystems, Buffalo Grove, IL, USA, 1/1000) or N-terminal end (Romeo, JAI-1-49-3, ABCAm, Cambridge, MA, USA, 1/500), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (10R-G109a Fitzgerald, Acton, MA, USA, 1/1000). Blots were quantitatively analyzed using a LI-COR Odyssey infrared imager.