Immunofluorescent Labeling of Muscle Tissues

The gastrocnemius, plantaris, and soleus were removed intact from mice, flash-frozen in liquid nitrogen–cooled isopentane, and cryosectioned (7 μm). Before labeling, sections were fixed with 0.5% paraformaldehyde, permeabilized with 0.5% Triton X-100/PBS/0.2 M NH₄Cl, and blocked with PBS containing 2% fish gelatin/0.08% BSA. Sections were incubated with primary antibodies (30 nM). After several PBS washes, sections were incubated with a cocktail containing either goat anti–rabbit IgG or anti–mouse IgM conjugated to Texas red (Jackson Immunoresearch; 1:200) mixed with either donkey anti–mouse IgG conjugated FITC or α-bungarotoxin (Tx) conjugated to BODIPY-fluorescence (Molecular Probes, Eugene, OR; 1:300). Washed sections were fixed with 4% paraformaldehyde and mounted in glycerol containing n-propyl gallate to reduce photobleaching (20 (link)). Antibody specificity was tested by preincubating antibodies with their antigenic peptide (100 μM) for 30 min before labeling. Adjacent sections were stained with Mayers haematoxylin-eosin (30 ). Sections were viewed on a fluorescence microscope (model Axioskop; Carl Zeiss, Inc., Thornwood, NY) and photographed (TMax 400 film; Kodak, Rochester, NY).