Tissues from muscle biopsy specimens were frozen and stored at -80°C until analysis. Protein isolation and Western blot procedures were performed as described previously.20 (link) Transferred proteins were detected with antibodies against NEFL (PA1-32240, 1:250 dilution; Thermo Scientific) and GAPDH (FL-335, 1:1000 dilution; Santa Cruz Biotechnology) and visualized using enhanced chemiluminescence. Quantification of protein levels was normalized to GAPDH using a software program (Quantity One 4.2.1; Bio-Rad) on an image station (440 Kodak DS; Eastman Kodak Co).
For immunofluorescence studies, 8-μm frozen transverse sections of muscle biopsy specimens from patients and age-matched controls were stained with rabbit polyclonal anti-NEFL (PA1-32240, 1:250 dilution; Thermo Scientific) and a-bun-garotoxin AlexaFluor 594 conjugate (B13423, 1:40 dilution; Invitrogen) antibodies. Alexa Fluor-conjugated antirabbit IgG (1: 100; Molecular Probes) was used as a secondary antibody. Coverslips were mounted using mounting medium with 4′,6-diamidino-2-phenylindole dihydrochloride (Vectashield; Vector Laboratories). Staining was evaluated using a microscope (Eclipse 90i; Nikon) with software (NIS-Elements AR; Nikon).
For immunofluorescence studies, 8-μm frozen transverse sections of muscle biopsy specimens from patients and age-matched controls were stained with rabbit polyclonal anti-NEFL (PA1-32240, 1:250 dilution; Thermo Scientific) and a-bun-garotoxin AlexaFluor 594 conjugate (B13423, 1:40 dilution; Invitrogen) antibodies. Alexa Fluor-conjugated antirabbit IgG (1: 100; Molecular Probes) was used as a secondary antibody. Coverslips were mounted using mounting medium with 4′,6-diamidino-2-phenylindole dihydrochloride (Vectashield; Vector Laboratories). Staining was evaluated using a microscope (Eclipse 90i; Nikon) with software (NIS-Elements AR; Nikon).