Alexa fluor conjugated anti rabbit igg

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).

B220⁺IgG1⁺CD38⁺NP⁺ memory B cells were added to slides by cytospin. The cells were fixed, incubated with rabbit anti-LC3 (Abgent), followed by staining with Alexa Fluor-conjugated anti-rabbit IgG (Molecular Probes). The nucleus was counter-stained with DAPI. The cells were then analyzed using a SoftWorx Image deconvolution microscope (Applied Precision).

For mouse studies, cryo-sections (7 µm) of frozen muscles were processed for hematoxylin and eosin (HE) and NADH-TR stainings using standard procedures. The proportion of internalized nuclei and the diameter of myofibers were quantified as previously described (16 (link)). For canine studies, serial 8 µm thick transverse cryo-sections were prepared from frozen muscles and processed for hematoxylin and eosin (HE) and NADH-TR stainings. The number of centrally nucleated fibers, and fibers with mislocalization of organelles (including mitochondrial aggregates and necklace fibers) were quantified manually from photographs taken at 200x magnification. For fiber size quantification, muscles were immunostained as described above with rabbit anti-dystrophin antibodies (Abcam PLC, ab15277) and AlexaFluor-conjugated anti-rabbit IgG (Molecular Probes). Staining was evaluated and MinFeret diameters of fibers were quantified using a Nikon Eclipse 90i microscope using NIS-Elements AR software (Nikon Instruments Inc.) and a BX53 microscope and CellSens Standard software (Olympus). Slides for evaluation of canine cardiac pathology were produced through the Children’s Research Institute Histology Core Facility at MCW. Electron microscopy was performed at MCW’s Electron Microscopy Core Facility and quantification of sarcotubular structures was performed as described previously (35 (link)).