Quantitative Analysis of Immunofluorescent Signals for Dystrophin, β-Dystroglycan and Myosin in Skeletal Muscle by Epifluorescence Microscopy

Quantitative analysis of signal intensities in immunostained sections has been performed in only a few studies owing to difficulties with quantifying amounts of antigen present. We determined correlations between fluorescent signal intensities and amounts of antigen in muscle cryosections by altering section thickness from 4 to 10 μm. Fluorescent signals of dystrophin. β-dystroglycan and myosin were detected with monoclonal and/or poly-clonal primary antibodies using routine procedures. Confocal laser microscopy demonstrated that these signals were distributed uniformly along the z-axis suggesting that the antibodies permeated well through the sections. Epifluorescence microscopy with microfluor-ometry demonstrated a positive correlation between the optical density of signals and section thickness. These findings suggest that immunofluorescent signals can be quantitated by epifluorescence microscopy.